RU2788083C2 - Peptide for treatment of age-related macular degeneration - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, and it is mutant SPINK2 peptide, which inhibits protease activity of human HTRA1. The invention also relates to polynucleotide for use in the treatment or prevention of a disease related to HTRA1, containing a nucleotide sequence encoding an amino acid sequence contained in the obtained peptide, as well as to the use of such a peptide as part of vectors, conjugates, pharmaceutical compositions for use in the treatment or prevention of a disease related to HTRA.

EFFECT: invention allows for an increase in the efficiency of the treatment of diseases related to HTRA.

30 cl, 77 dwg, 2 tbl, 11 ex

Description

The field of technology to which the invention belongs

[0001] The present invention relates to a peptide, a polynucleotide, a vector, a cell, a method for producing a peptide, a peptide obtained by this method, a composition containing the peptide, a pharmaceutical composition containing the peptide, a pharmaceutical composition for the treatment or prevention of various diseases containing the peptide, the use of the peptide for treating or preventing various diseases, a method for treating various diseases, including the step of administering a peptide, and so on.

State of the art

[0002] Heat shock A inducible serine peptidase 1 (HTRA1) is a trypsin-like serine protease (PRSS11; PA clan, family S1) and consists of an N-terminal domain including an IGFBP-like motif and a Kazal-like motif, a protease domain, and a C -terminal PDZ domain. HTRA1 belongs to the HTRA family, including HTRA2, HTRA3 and HTRA4, and reversibly has active and inactive structures in the same way as other HTRA molecules (non-patent documents 1 and 2). Its expression is unevenly distributed in the human body and is found at relatively high levels in cartilage, synovium, placenta, and the like. HTRA1 is known to cleave many extracellular matrix components such as amyloid precursor protein, fibromodulin, clusterin, ADAM9 and vitronectin as substrates, and is associated with diseases such as arthritis and bone calcification (Non-Patent Documents 3, 4, 5 and 6). It is also known that if the HTRA1 promoter region has a genetic polymorphism (rs11200638), then the level of HTRA1 transcription is increased. The genome-wide association analysis also revealed that the polymorphism is highly correlated with age-related macular degeneration (hereinafter referred to as "AMD") (Non-Patent Documents 7 and 8).

AMD is a chronic degenerative disease with aging and is characterized by loss of central vision. This disease is the leading cause of acquired blindness in the United States and is the fourth most common cause of acquired blindness after glaucoma, diabetic retinopathy, and retinitis pigmentosa in Japan (Non-Patent Document 12). HTRA1 mRNA and protein levels are elevated in lymphocytes or retinal pigment epithelial cells in AMD patients (Non-Patent Document 13). It has also been reported that HTRA1 protein expression is increased in optic disc drusen cells or degenerated retinal pigment epithelial cells, which are precursors of AMD lesions or neovascular membranes (Non-Patent Documents 11 and 14-16). It has also been reported that the HTRA1 protein is found in the vitreous in association with retinal detachment, retinal vein occlusion, vitreous hemorrhage, macular hole, and the like, and its amount synchronizes with the angiogenesis marker VEGF (Non-Patent Document 17). In addition, cleavage of proteins constituting a basement membrane such as fibulin 5 or tropoelastin and fragmentation of the elastic layer of Bruch's membrane were observed in HTRA1 transgenic mice (Non-Patent Document 9). However, it has not been directly shown that inhibition of HTRA1 protease activity is effective or ineffective for the treatment of the diseases described above and for the protection of the retina.

[0003] SPINK2 (Kazal-type 2 serine protease inhibitor) is a Kazal-like domain containing three disulfide bonds and functioning as a trypsin/acrosin inhibitor (Non-Patent Document 10). However, its association with AMD has not yet been clarified.

Mouse and rabbit models are known as animal models for AMD research (Non-Patent Documents 18, 19 and 20). With regard to rabbits, which are more preferable as models due to the fact that the results obtained on them can be extrapolated to human eye diseases (Non-Patent Document 21), conventional models are used simply to observe abnormal phenomena in the retina and choroid, and there are difficulties in assessing abnormal functions of retinal pigment epithelial cells (RPE cells), etc. Another problem is the long period of up to 8 months required for model formation (non-patent document 20).

Link List

Non-Patent Documents

[0004] Non-Patent Document 1: Truebestein L. et al., 2011, Nat. Struct. Mol. Biol., Vol. 18 (No. 3): p. 386-8

Non-Patent Document 2: Eigenbrot C. et al., 2012, Structure, Vol. 20 (No. 6): p. 1040-50

Non-Patent Document 3: Grau S. et al., 2005, Proc. Natl. Acad. sci. USA., Vol. 102 (No. 17): p. 6021-26

Non-Patent Document 4: Grau S. et al., 2006, J. Biol. Chem., Vol. 281 (No. 10): p. 6124-29

Non-Patent Document 5: Hadfield K.D. et al., 2008, J. Biol. Chem., Vol. 283, (No. 9): p. 5928-38

Non-Patent Document 6: An E. et al., 2010, Invest. Ophthalmol. Vis. Sc., Vol. 51 (No. 7): p. 3379-86

Non-Patent Document 7: Yang Z. et al., 2006, Science, Vol. 314 (No. 5801): p. 992-93

Non-Patent Document 8: Tang N.P. et al., 2009, Ann. Epidemiol., Vol. 19 (No. 10): p. 740-45

Non-Patent Document 9: Vierkotten S. et al., 2011, PLoS One, Vol. 6 (No. 8): p. e22959

Non-Patent Document 10: Chen T, et al., 2009, Proteins, Vol. 77 (No. 1): p. 209-19

Non-Patent Document 11: Yang Z. et al., Science, 2006, Vol. 314, no. 5801: p. 992-93

Non-Patent Document 12: Kimihiro Nakae et al., 2007 Annual report of the Research Committee on Chorioretinal Degenerations and Optic Atrophy, Research on Measures for Intractable Diseases, the Ministry of Health, Labor and Welfare of Japan (2007)

Non-Patent Document 13: Black J.R. and Clark S.J., Genet. Med., 2016, Vol. 18, no. 4: p. 283-89

Non-Patent Document 14: Cameron D.J. et al., Cell Cycle, 2007, Vol. 6, no. 9: p. 1122-25

Non-Patent Document 15: Chan C.C. et al., Trans. Am. Soc., 2007, Vol. 105: p. 92-97

Non-Patent Document 16: Tuo J. et al., 2008, Vol. 115, no. 11: p. 1891-98

Non-Patent Document 17: Ng TK et al., Invest. Ophthalmol. Vis. Sc., 2011, Vol. 52, no. 6: p. 3706-12

Non-Patent Document 18: Espinosa-Haidemann D.G et al., Investigative Ophthalmology & Visual Science, 2006, Vol. 47 (No. 2): p. 729-37

Non-Patent Document 19: Pons M. et al., American Journal of Pathology, 2010, Vol. 177: p. 1198-1213

Non-Patent Document 20: Trivino A. et al., Experimental Eye Research, 2006, Vol. 83: p. 357-366

Non-Patent Document 21: Zernii E.Y. et al., CNS & Neurological Disorders-Drug Targets, 2016, Vol. 15: p. 267-291.

The essence of the invention

Technical task

[0005] It is an object of the present invention to provide a novel inhibitor of heat shock inducible serine peptidase A 1 (HTRA1).

The solution of the problem

[0006]

The present invention provides the following:

(1) A mutant SPINK2 peptide that contains the amino acid sequence shown in SEQ ID NO: 30 (FIG. 42) and inhibits the protease activity of human HTRA1;

(2) The peptide according to (1), wherein the first Xaa (X ₁ ) is Asp, Glu, Ser, Gly or Ile, the second Xaa (X ₂ ) is Ala, Gly, Leu, Ser or Thr, the third Xaa (X ₃ ) represents Asp, His, Lys, Met or Gln, the fourth Xaa (X ₄ ) represents Asp, Phe, His, Ser or Tyr, the fifth Xaa (X ₅ ) represents Ala, Asp, Glu, Met or Asn, the sixth Xaa ( X ₆ ) represents Met or Trp, the seventh Xaa (X ₇ ) represents Gln, Trp, Tyr or Val, the eighth Xaa (X ₈ ) represents Phe, Leu or Tyr, the ninth Xaa (X ₉ ) represents Phe or Tyr, the tenth Xaa ( X ₁₀ ) represents Ala, Glu, Met or Val, and the eleventh Xaa (X ₁₁ ) represents Ala, Thr or Val;

(3) The peptide according to (1) or (2), which contains the amino acid sequence shown in any of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23 -29 (figs. 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 and 35-41);

(4) The peptide according to any one of paragraphs (1)-(3), which contains an amino acid sequence obtained by linking a peptide bond of one to three amino acids with the amino-terminal portion of the amino acid sequence shown in SEQ ID NO: 30 (Fig. 42);

(5) A peptide according to any one of paragraphs (1) to (4), which contains an amino acid sequence obtained by linking a peptide bond of one or two amino acids with the carboxy-terminal portion of the amino acid sequence shown in SEQ ID NO: 30 (Fig. 42);

(6) The peptide according to any one of paragraphs (1) to (5), which has a three-dimensional structure, characterized by the presence of three disulfide bonds and includes a loop structure, α-helix and β-sheet;

(7) Polynucleotide containing a nucleotide sequence encoding the amino acid sequence contained in the peptide according to any one of paragraphs (1)-(6);

(8) A vector containing a polynucleotide according to (7);

(9) A cell containing a polynucleotide according to paragraph (7) or a vector according to paragraph (8) or producing a peptide according to any one of paragraphs (1) to (6);

(10) A method for producing a mutant SPINK2 peptide that inhibits HTRA1 protease activity, comprising the following steps (i) and (ii):

(i) culturing the cell according to item (9); and

(ii) isolating the mutant SPINK2 peptide from the culture;

(11) Mutant SPINK2 peptide obtained by the method of (10);

(12) A conjugate containing a peptide according to any one of paragraphs (1)-(6) and (11) associated with an additional molecule;

(13) The conjugate according to (12), which is a polypeptide;

(14) An antibody that binds to the peptide according to any one of paragraphs (1)-(6) and (11), or a functional fragment thereof;

(15) A composition comprising a peptide according to any one of paragraphs (1) to (6) and (11), a polynucleotide according to paragraph (7), a vector according to paragraph (8), a cell according to paragraph (9), a conjugate according to items (12) or (13) and/or an antibody or a functional fragment thereof according to item (14);

(16) A pharmaceutical composition containing a peptide according to any one of paragraphs. (1)-(6) and (11) and/or the conjugate according to paragraphs. (12) or (13);

(17) The pharmaceutical composition according to (16) for the treatment or prevention of a disease associated with HTRA1;

(18) The pharmaceutical composition according to (17), wherein the disease associated with HTRA1 is one, two or more diseases selected from the group consisting of wet age-related macular degeneration, dry age-related macular degeneration, geographic atrophy, diabetic retinopathy , retinopathy of prematurity, polypoid choroidal vasculopathy, rheumatoid arthritis and osteoarthritis;

(19) The pharmaceutical composition according to any one of paragraphs. (16)-(18) which contains one, two or more additional drugs;

(20) The pharmaceutical composition according to any one of paragraphs. (16)-(18), which is used in combination with one, two or more additional drugs;

(21) The pharmaceutical composition according to any one of paragraphs. (16)-(20) which is a retinal protection agent;

(22) A method for identifying a therapeutic agent or prophylactic agent for age-related macular degeneration, comprising the following steps 1-3:

[step 1] incubation of HTRA1 protease and substrate in the presence and absence of the test substance;

[step 2] detecting HTRA1 protease activity in the presence and absence of the test substance; and

[step 3] determining the test substance as positive when the activity of the HTRA1 protease in the presence of the test substance is lower than the activity of the HTRA1 protease in the absence of the test substance;

(23) A method for identifying an agent for protecting the retina, including the following steps 1-3:

[step 1] incubation of HTRA1 protease and substrate in the presence and absence of the test substance;

[step 2] detecting HTRA1 protease activity in the presence and absence of the test substance; and

[step 3] determining the test substance as positive when the activity of the HTRA1 protease in the presence of the test substance is lower than the activity of the HTRA1 protease in the absence of the test substance;

(24) A method for producing a rabbit retinal injury model, comprising the step of feeding rabbits a high-fat diet including hydroquinone for 3-6 months, wherein the model rabbit's retinal pigment epithelium cells are hypertrophied compared to normal rabbit's retinal pigment epithelial cells;

(25) The rabbit retinal injury model obtained by the method of (24), wherein the rabbit retinal injury model has hypertrophied retinal pigment epithelial cells compared to a normal rabbit;

(26) A method for identifying a therapeutic agent or prophylactic agent for age-related macular degeneration or a retinal protective agent, comprising the steps of (i) and (ii):

(i) evaluating hypertrophy of retinal pigment epithelial cells in a rabbit according to (25) with and without administration of the test substance; and

(ii) defining a test substance as positive when retinal pigment epithelial cell hypertrophy is suppressed when the test substance is administered compared to retinal pigment epithelial cell hypertrophy without the test substance;

(27) The method according to (26), wherein the evaluation in step (i) is a measurement of the mean area of retinal pigment epithelium cells or the number of hypertrophied retinal pigment epithelium cells;

(28) The pharmaceutical composition according to any one of paragraphs. (16)-(18) for the treatment or prevention of the dry form of age-related macular degeneration; and

(29) The pharmaceutical composition according to any one of paragraphs. (16)-(18) for the treatment or prevention of the wet form of age-related macular degeneration; etc.

Advantageous Effects of the Invention

[0007] The peptide provided by the present invention and the pharmaceutical composition containing the peptide have HTRA1 inhibitory activity, and are useful in treatment or prevention, etc. age-related macular degeneration.

Brief description of the figures

[0008] [Fig. 1(A)] In FIG. 1(A) is a diagram showing the results of comparing human, mouse, rat and monkey HTRA1 sequence similarity. The dotted line shows the enzymatically active domain (from 204Gly to 364Leu).

[Fig. 1(B)] In FIG. 1(B) is a diagram showing the results of comparing human, mouse, rat and monkey HTRA1 sequence similarity (continued).

[Fig. 2] In FIG. 2 is a graph showing the results of evaluating the HTRA1 inhibitory activity (catalyte) of an HTRA1 inhibitory peptide using the cleavage rate of the peptide substrate as an index. Panels A-C show the results of the evaluation for each inhibitory peptide, and panel D shows the results of the evaluation of the wild-type control SPINK2.

[Fig. 3] In FIG. 3 is a graph showing the results of evaluating the inhibition activity of HTRA1 (total), HTRA1 inhibitory peptide using the cleavage rate of the peptide substrate as an index (panels A-C).

[Fig. 4(A)] In FIG. 4(A) is a graph showing the results of evaluating the HTRA1 inhibitory activity (catalyte) of an HTRA1 inhibitory peptide using human vitronectin cleavage as an index. Analysis was performed by Western blotting using an antibody to human vitronectin (R&D Systems, Inc.; MAB2349).

[Fig. 4(B)] In FIG. 4(B) is a graph showing the results of evaluating the HTRA1 inhibitory activity (catalyte) of an HTRA1 inhibitory peptide using human vitronectin cleavage as an index (continued).

[Fig. 5(A)] In FIG. 5(A) is a graph showing the results of an evaluation of the cross-reactivity of an HTRA1 inhibitory peptide with each protease using the cleavage of the peptide substrate as an indicator (Part 1). The name of each protease used and its concentration, and the name of the substrate and its concentration, etc. see example 3.

[Fig. 5(B)] In FIG. 5(B) is a graph showing the results of an evaluation of the cross-reactivity of an HTRA1 inhibitory peptide with each protease using the cleavage of the peptide substrate as an index (Part 2).

[Fig. 5(C)] FIG. 5(C) is a graph showing the results of an evaluation of the cross-reactivity of an HTRA1 inhibitory peptide with each protease using peptide substrate cleavage as an index (part 3).

[Fig. 5(D)] In FIG. 5(D) is a graph showing the results of an evaluation of the cross-reactivity of an HTRA1 inhibitory peptide with each protease using the cleavage of the peptide substrate as an index (part 4).

[Fig. 6] In FIG. 6 is a diagram showing the HTRA1 (catalyte)/HTRA1 inhibitory peptide complex obtained by X-ray crystallography. The inhibitory peptide is associated with each molecule of the HTRA1 trimer formed by HTRA1 (catalyte).

[Fig. 7] In FIG. 7 is a diagram showing the HTRA1 (catalyte)/HTRA1 inhibitory peptide complex obtained by X-ray crystallography as a monomer. The inhibitory peptide is associated with the region containing the active site of HTRA1 (catalyte).

[Fig. 8] FIG. 8 shows the amino acid sequence of (SEQ ID NO: 54) H2-Opt. The N-terminal "Mca-I" is N-(4-methylcoumaryl-7-amide)isoleucine and the C-terminal "(Dnp)K" is N-epsilon-(2,4-dinitrophenyl)lysine.

[Fig. 9] FIG. 9 is a diagram showing that HTRA1 expression was upregulated in the vitreous of a rat model of light-induced retinal injury. Analysis was performed by Western blot using anti-human HTRA1/PRSS11 antibody (R&D Systems, Inc.; AF2916).

[Fig. 10] FIG. 10 is a graph showing that in the HTRA1 inhibitory peptide administration group in the rat model of light-induced retinal injury, the decrease in the number of nuclei in the outer nuclear layer in the retinal cross section was suppressed. n=4 for the saline group and n=5 for other groups.

[Fig. 11] In FIG. 11 is a graph showing the results of evaluating the HTRA1 inhibitory activity (catalyte) of five HTRA1 inhibitory peptide derivatives using the cleavage rate of the peptide substrate as an index.

[Fig. 12] FIG. 12 is a graph showing the results of binding of three HTRA1 inhibitory peptides to HTRA1 (catalyte) by immunoprecipitation.

[Fig. 13] FIG. 13 shows the amino acid sequence (SEQ ID NO: 1) of human SPINK2.

[Fig. 14] FIG. 14 shows the nucleotide sequence (SEQ ID NO: 2) encoding the amino acid sequence of human SPINK2.

[Fig. 15] FIG. 15 shows the amino acid sequence (SEQ ID NO: 3) of the H218 peptide.

[Fig. 16] FIG. 16 shows the nucleotide sequence (SEQ ID NO: 4) encoding the amino acid sequence of the H218 peptide.

[Fig. 17] FIG. 17 shows the amino acid sequence (SEQ ID NO: 5) of the H223 peptide.

[Fig. 18] FIG. 18 shows the nucleotide sequence (SEQ ID NO: 6) encoding the amino acid sequence of the H223 peptide.

[Fig. 19] FIG. 19 shows the amino acid sequence (SEQ ID NO: 7) of the H228 peptide.

[Fig. 20] FIG. 20 shows the nucleotide sequence (SEQ ID NO: 8) encoding the amino acid sequence of the H228 peptide.

[Fig. 21] FIG. 21 shows the amino acid sequence (SEQ ID NO: 9) of the H308 peptide.

[Fig. 22] FIG. 22 shows the nucleotide sequence (SEQ ID NO: 10) encoding the amino acid sequence of the H308 peptide.

[Fig. 23] In FIG. 23 shows the amino acid sequence (SEQ ID NO: 11) of the H321 peptide.

[Fig. 24] FIG. 24 shows the nucleotide sequence (SEQ ID NO: 12) encoding the amino acid sequence of the H321 peptide.

[Fig. 25] FIG. 25 shows the amino acid sequence (SEQ ID NO: 13) of the H322 peptide.

[Fig. 26] FIG. 26 shows the nucleotide sequence (SEQ ID NO: 14) encoding the amino acid sequence of the H322 peptide.

[Fig. 27] FIG. 27 shows the amino acid sequence (SEQ ID NO: 15) of a peptide derivative of H308AT.

[Fig. 28] FIG. 26 shows the nucleotide sequence (SEQ ID NO: 16) encoding the amino acid sequence of the H308AT peptide derivative.

[Fig. 29] FIG. 29 shows the amino acid sequence (SEQ ID NO: 17) of the H321AT peptide derivative.

[Fig. 30] FIG. 30 shows the nucleotide sequence (SEQ ID NO: 18) encoding the amino acid sequence of the H321AT peptide derivative.

[Fig. 31] In FIG. 31 shows the amino acid sequence (SEQ ID NO: 19) of a peptide derivative of H322AT.

[Fig. 32] FIG. 32 shows the nucleotide sequence (SEQ ID NO: 20) encoding the amino acid sequence of the H322AT peptide derivative.

[Fig. 33] In FIG. 33 shows the amino acid sequence (SEQ ID NO: 21) of the M7 peptide.

[Fig. 34] FIG. 34 shows the nucleotide sequence (SEQ ID NO: 22) encoding the amino acid sequence of the M7 peptide.

[Fig. 35] FIG. 35 shows the amino acid sequence (SEQ ID NO: 23) of the H308_S16A peptide derivative.

[Fig. 36] FIG. 36 shows the amino acid sequence (SEQ ID NO: 24) of the peptide derivative H308_D1G_S16A.

[Fig. 37] FIG. 37 shows the amino acid sequence (SEQ ID NO: 25) of the peptide derivative H308_D1S_S16A.

[Fig. 38] FIG. 38 shows the amino acid sequence (SEQ ID NO: 26) of the peptide derivative H308_D1E_S16A.

[Fig. 39] FIG. 39 shows the amino acid sequence (SEQ ID NO: 27) of the peptide derivative H308_D1SLI_S16A.

[Fig. 40] In FIG. 40 shows the amino acid sequence (SEQ ID NO: 28) of a peptide derivative of H321ATS16A.

[Fig. 41] In FIG. 41 shows the amino acid sequence (SEQ ID NO: 29) of a peptide derivative of H322ATS16A.

[Fig. 42] In FIG. 42 shows the general formula (SEQ ID NO: 30) of an HTRA1 inhibitory peptide. X ₁ -X ₁₁ each represents an arbitrary amino acid.

[Fig. 43] In FIG. 43 shows the amino acid sequence (SEQ ID NO: 31) consisting of an S-tag and a linker.

[Fig. 44] In FIG. 44 shows the amino acid sequence (SEQ ID NO: 32) of the C-terminal hexamer.

[Fig. 45] In FIG. 45 shows the nucleotide sequence (SEQ ID NO: 33) of primer 1.

[Fig. 46] FIG. 46 shows the nucleotide sequence (SEQ ID NO: 34) of primer 2.

[Fig. 47] In FIG. 47 shows the nucleotide sequence (SEQ ID NO: 35) of primer 3.

[Fig. 48] FIG. 48 shows the nucleotide sequence (SEQ ID NO: 36) of primer 4.

[Fig. 49] In FIG. 49 shows the nucleotide sequence (SEQ ID NO: 37) of primer 5.

[Fig. 50] FIG. 50 shows the nucleotide sequence (SEQ ID NO: 38) of primer 6.

[Fig. 51] In FIG. 51 shows the nucleotide sequence (SEQ ID NO: 39) of primer 7.

[Fig. 52] In FIG. 52 shows the nucleotide sequence (SEQ ID NO: 40) of primer 8.

[Fig. 53] In FIG. 53 shows the nucleotide sequence (SEQ ID NO: 41) of primer 9.

[Fig. 54] In FIG. 54 shows the nucleotide sequence (SEQ ID NO: 42) of primer 10.

[Fig. 55] In FIG. 55 shows the nucleotide sequence (SEQ ID NO: 43) of primer 11.

[Fig. 56] In FIG. 56 shows the nucleotide sequence (SEQ ID NO: 44) of primer 12.

[Fig. 57] FIG. 57 shows the nucleotide sequence (SEQ ID NO: 45) of primer 13.

[Fig. 58] FIG. 58 shows the nucleotide sequence (SEQ ID NO: 46) of primer 14.

[Fig. 59] FIG. 59 shows the nucleotide sequence (SEQ ID NO: 47) of primer 15.

[Fig. 60] FIG. 60 shows the nucleotide sequence (SEQ ID NO: 48) of primer 16.

[Fig. 61] FIG. 61 shows the nucleotide sequence (SEQ ID NO: 49) of primer 17.

[Fig. 62] FIG. 62 shows the nucleotide sequence (SEQ ID NO: 50) of primer 18.

[Fig. 63] In FIG. 63 shows the nucleotide sequence (SEQ ID NO: 51) of primer 19.

[Fig. 64] FIG. 64 shows the nucleotide sequence (SEQ ID NO: 52) of primer 20.

[Fig. 65] In FIG. 65 shows the nucleotide sequence (SEQ ID NO: 53) of human HTRA1 (complete).

[Fig. 66(A)] In FIG. 66(A) is a graph showing the results of evaluating the HTRA1 inhibition activity (catalyte) of HTRA1 inhibitory peptides using the cleavage rate of the peptide substrate as an index.

[Fig. 66(B)] In FIG. 66(B) is a graph showing the results of evaluating the HTRA1 inhibition activity (catalyte) of an HTRA1 inhibitory peptide using the cleavage rate of the peptide substrate as an index.

[Fig. 67] FIG. 67 is a graph showing the results of evaluating the HTRA1 inhibition activity (catalyte) of an HTRA1 inhibitory peptide using human vitronectin cleavage as an index. Analysis was performed by Western blotting using an antibody to human vitronectin (R&D Systems, Inc.; MAB2349).

[Fig. 68(A)] FIG. 68(A) are graphs showing the results of an evaluation of the cross-reactivity of HTRA1 inhibitory peptides with each protease using peptide substrate cleavage as an index (Part 1).

[Fig. 68(B)] In FIG. 68(B) is a graph showing the results of an evaluation of the cross-reactivity of an HTRA1 inhibitory peptide with each protease using the cleavage of the peptide substrate as an indicator (Part 2).

[Fig. 68(C)] FIG. 68(C) are graphs showing the results of an evaluation of the cross-reactivity of an HTRA1 inhibitory peptide with each protease using peptide substrate cleavage as an index (part 3).

[Fig. 68(D)] In FIG. 68(D) are graphs showing the results of an evaluation of the cross-reactivity of an HTRA1 inhibitory peptide with each protease using peptide substrate cleavage as an index (part 4).

[Fig. 68(E)] In FIG. 68(E) are graphs showing the results of an evaluation of the cross-reactivity of an HTRA1 inhibitory peptide with each protease using peptide substrate cleavage as an index (part 5).

[Fig. 69] FIG. 69 is a graph showing the results of binding of three HTRA1 inhibitory peptides to HTRA1 (catalyte) by immunoprecipitation.

[Fig. 70(A)] In FIG. 70(A) is a graph showing that the HTRA1 inhibitory peptide H308_D1G_S16A administration group in the rat model of light-induced retinal injury suppressed the reduction in the number of nuclei in the outer nuclear layer in the retinal cross section. n=6 for all groups. The dose of HTRA1 inhibitory peptide H308_D1G_S16A was 0.2 and 1 μg/eye.

[Fig. 70(B)] In FIG. 70(B) is a graph showing that in the HTRA1 inhibitory peptide H321AT_D1G_S16A administration group in the rat model of light-induced retinal injury, the decrease in the number of nuclei in the outer nuclear layer in the retinal cross section was suppressed. n=6 for all groups. The dose of HTRA1 inhibitory peptide H321AT_D1G_S16A was 0.2 and 1 μg/eye.

[Fig. 70(C)] In FIG. 70(C) is a graph showing that the HTRA1 inhibitory peptide H322AT_D1G_S16A administration group in the rat model of light-induced retinal injury suppressed the reduction in the number of nuclei in the outer nuclear layer in the retinal cross section. n=6 for all groups. The dose of HTRA1 inhibitory peptide H322AT_D1G_S16A was 0.2 and 1 μg/eye.

[Fig. 71(A)] In FIG. 71(A) is a graph showing the results of immunostaining of RPE cells in a 12-week-old rabbit, a 3-year-old rabbit, and a 3-year-old rabbit fed an HFD-HQ diet with ZO-1 antibody (Thermo Fisher Scientific Inc.).

[Fig. 71(B)] In FIG. 71(B) shows mean RPE cell areas in a 12-week-old rabbit, a 3-year-old rabbit, and a 3-year-old rabbit fed an HFD-HQ diet.

[Fig. 71(C)] In FIG. 71(C) is a graph showing increased mRNA expression of complement component 3 "C3", which is an AMD-associated factor, in the retinal tissue of a 3-year-old rabbit fed an HFD-HQ diet.

[Fig. 71(D)] In FIG. 71(D) is a graph showing increased mRNA expression of complement component 3 "C3", which is an AMD-associated factor, in RPE/choroid tissue of a 3-year-old rabbit fed an HFD-HQ diet.

[Fig. 71(E)] In FIG. 71(E) is a graph showing the results of measurement of the HTRA1 concentration in the vitreous of a 12-week-old rabbit, a 3-year-old rabbit, and a 3-year-old rabbit fed an HFD-HQ diet by LC-MS/MS.

[Fig. 72(A)] FIG. 72(A) is a graph showing that in the HTRA1 inhibitory peptide H308 group, 3-year-old rabbits fed the HFD-HQ diet showed an inhibitory effect on RPE cell hypertrophy. n=5 for all groups. The mean area was used as an indicator.

[Fig. 72(B)] In FIG. 72(B) is a graph showing that in the HTRA1 inhibitory peptide H308 group, 3-year-old rabbits fed the HFD-HQ diet showed an inhibitory effect on RPE cell hypertrophy. n=5 for all groups. The number of RPE cells having a cell area of 1500 m ² or more was used as an indicator.

[Fig. 73] In FIG. 73 is a diagram showing the results of comparison of human, monkey, rabbit, mouse, and rat HTRA1 sequence similarity. The dotted line represents the enzymatically active domain (204Gly to 364Leu).

[Fig. 74] FIG. 74 is a graph showing that an HTRA1 inhibitory peptide exhibited a suppressive effect on VEGF mRNA induced in the human retinal pigment epithelial cell line ARPE-19 by the addition of H ₂ O ₂ , normal human serum complement and HTRA1.

[Fig. 75] FIG. 75 is a graph showing that the HTRA1 inhibitory peptide had a suppressive effect on serum-induced human umbilical vein endothelial cell (HUVEC) migration.

[Fig. 76] FIG. 76 shows the nucleotide sequence of primer 21.

[Fig. 77] FIG. 77 shows the nucleotide sequence of primer 22.

In the present invention, the expression "SEQ ID NO: X (Fig. Y)" or "Fig. Y (SEQ ID NO: X)" means that the sequence is shown in SEQ ID NO: X or shown in FIG. Y.

Description of Embodiments

1. Definitions

[0009] In the present invention, the term "gene" is used to refer to a nucleic acid molecule containing a nucleotide sequence encoding an amino acid sequence contained in a protein, or its complementary chain. A gene consists of a single strand, a double strand, or a triple or more strand. The association of a DNA strand and an RNA strand, ribonucleotides and deoxyribonucleotides in the same strand, and a double-stranded or triple-stranded or longer nucleic acid molecule comprising such a strand is also included within the meaning of the term "gene".

[0010] In the present invention, the terms "gene", "polynucleotide" and "nucleic acid molecule" are synonymous with each other and are not limited to the number of their constituent units, such as ribonucleotides, deoxyribonucleotides, nucleotides and nucleosides, in any way. For example, DNA, RNA, mRNA, cDNA, cRNA, probe, oligonucleotide, primer, and the like are also included within the scope of these terms. The term "nucleic acid molecule" is also abbreviated as "nucleic acid".

[0011] In the present invention, the terms "polypeptide", "peptide" and "protein" are synonymous with each other. A peptide that inhibits or suppresses one, two or more activities or functions of the target molecule X (such inhibitory or inhibitory effects are collectively referred to hereinafter as "X inhibitory activity") may be referred to as an "X inhibitory peptide".

[0012] The term "SPINK2" is used to refer to a Kazal-type 2 serine protease inhibitor. This 7 kDa protein consists of a Kazal-like domain having three disulfide bonds. SPINK2 is preferably human-derived. In the present invention, the human SPINK2 is simply referred to as "SPINK2" unless otherwise indicated.

[0013] The term "HTRA1" is used to refer to heat shock A-induced serine peptidase 1. This protein consists of an N-terminal domain, including an IGFBP-like motif and a Kazal-like motif, a protease domain, and a C-terminal PDZ domain, and refers to HTRA family. HTRA1 is preferably human-derived. In the present invention, human HTRA1 is also simply referred to as "HTRA1" unless otherwise indicated.

[0014] The term "HTRA1 inhibitory peptide" is used to refer to a peptide that inhibits or suppresses one, two or more HTRA1 activities or functions. A peptide fragment, an adduct of a peptide with an additional molecule, or a peptide conjugate that retains HTRA1 inhibitory activity is included within the scope of the term "HTRA1 inhibitory peptide". In particular, a fragment, an adduct, and a modified form of a peptide that retains HTRA1 inhibitory activity are also included in the term "HTRA1 inhibitory peptide".

[0015] In the present invention, the term "cell" is used to refer to various cells derived from animals, subcultured cells, primary cultured cells, cell lines, recombinant cells, yeast, microorganisms, and the like.

[0016] In the present invention, the term "site" to which the peptide binds, i. A "site" that is recognized by a peptide is used to refer to a continuous or discontinuous incomplete amino acid sequence or incomplete conformation on a target molecule that binds or is recognized by a peptide. In the present invention, such a site may be referred to as an "epitope" or "binding site" on the target molecule.

[0017] The term "SPINK2 mutant" is used to refer to a peptide containing an amino acid sequence derived from the wild-type SPINK2 amino acid sequence by replacing one, two or more amino acids other than the amino acids in the wild-type sequence, deleting one, two or more amino acids from the wild-type sequence, insertion of one, two or more amino acids not present in the wild-type sequence, and/or adding amino acid(s) not present in the wild-type sequence to the amino-terminus (N-terminus) and/or carboxy-terminus (C-terminus) wild-type sequences (collectively referred to hereinafter as "mutations"). "SPINK2 mutant" having HTRA1 inhibitory activity is included in the term "HTRA1 inhibitory peptide". In the present invention, "insertion" can also be included in the term "addition".

[0018] In the present invention, the term "multiple" in the expression "one or more" refers to 3-10.

[0019] In the present invention, the expression "hybridize under stringent conditions" is used to mean that the hybridization is carried out under conditions that allow hybridization to be carried out at 65°C in a solution containing 5x SSC, and then the resulting product is washed at 65°C for 20 min in an aqueous solution containing 2x SSC C and 0.1% SDS at 65°C for 20 min in an aqueous solution containing 0.5x SSC and 0.1% SDS and at 65°C for 20 min in an aqueous solution containing 0.2x SSC and 0.1% SDS, or under conditions equivalent to those indicated. SSC means an aqueous solution of 150 mm NaCl and 15 mm sodium citrate, and n × SSC means n-fold concentration of SSC.

[0020] In the present invention, the terms "specific" and "specificity" are synonymous and interchangeable with the terms "selective" and "selectivity", respectively. For example, HTRA1-specific inhibitory peptide is synonymous with HTRA1-selective inhibitory peptide.

[0021] 2. Peptide

[0022] 2-1. Amino acid

The term "amino acid" is used to refer to an organic compound containing an amino group and a carboxyl group, and to refer to an α-amino acid contained as a constituent unit, preferably in a protein, and more preferably in a natural protein. In the present invention, the amino acid is more preferably Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val. The term "amino acid" is used to refer to these 20 amino acids in general, unless otherwise indicated. These 20 amino acids can be collectively referred to as "natural amino acids". The HTRA1 inhibitory peptide of the present invention preferably contains natural amino acids.

[0023] In the present invention, the term "amino acid residue" also refers to the term "amino acid".

[0024] In the present invention, an amino acid may be an L-amino acid, a D-amino acid, or a mixture thereof (DL-amino acid), and is an L-amino acid unless otherwise indicated.

[0025] Natural amino acids can be divided, for example, into the following groups, based on the general properties of the side chains:

(1) a group of hydrophobic amino acids: Met, Ala, Val, Leu and Ile;

(2) a group of neutral hydrophilic amino acids: Cys, Ser, Thr, Asn and Gln;

(3) a group of acidic amino acids: Asp and Glu;

(4) a group of basic amino acids: His, Lys and Arg;

(5) a group of amino acids that influence the direction of the main chain: Gly and Pro; and

(6) a group of aromatic amino acids: Trp, Tyr and Phe.

However, the classification of natural amino acids is not limited to this.

[0026] In the present invention, each naturally occurring amino acid may be subjected to a conservative amino acid substitution.

[0027] "Conservative amino acid substitution" means a substitution with a functionally equivalent or similar amino acid. A conservative amino acid substitution in a peptide causes a roughly static change in the amino acid sequence of the peptide. For example, one, two or more amino acid substitutions having similar polarity act functionally equivalent and cause a static change in the amino acid sequence of the peptide. In general, a substitution in a particular group can be considered as a conservative substitution in terms of structures and functions. However, as will be appreciated by those skilled in the art, the role of a particular amino acid residue can be determined by its position in the three-dimensional structure of the molecule containing the amino acid. For example, a cysteine residue may take an oxidized (disulfide) form having a lower polarity than a reduced (thiol) form. The long aliphatic side chain group of arginine is capable of defining structurally and functionally important features. Alternatively, a side chain containing an aromatic ring (tryptophan, tyrosine, and phenylalanine) is capable of contributing to an aromatic ion or cation-pi interaction. In such a case, even the replacement of amino acids having these side chains with amino acids belonging to the group of acidic or non-polar amino acids can be structurally and functionally conserved. Residues such as proline, glycine and cysteine (disulfide form) can have a direct effect on the three-dimensional structure of the backbone and often cannot be replaced without structural deformation.

[0028] A conservative amino acid substitution includes, as shown below, a specific substitution based on side chain similarity (L. Lehninger, Biochemistry, 2nd edition, pp. 73-75, Worth Publisher, New York (1975)), and a typical substitution.

(1) A group of non-polar amino acids: alanine (hereinafter referred to as "Ala" or simply "A"), valine (hereinafter referred to as "Val" or simply "V"), leucine (hereinafter referred to as "Leu" or simply "L" ), isoleucine (hereinafter referred to as "Ile" or simply "I"), proline (hereinafter referred to as "Pro" or simply "P"), phenylalanine (hereinafter referred to as "Phe" or simply "F"), tryptophan (hereinafter referred to as hereinafter "Trp" or simply "W") and methionine (hereinafter referred to as "Met" or simply "M");

(2) A group of uncharged polar amino acids: glycine (hereinafter referred to as "Gly" or simply "G"), serine (hereinafter referred to as "Ser" or simply "S"), threonine (hereinafter referred to as "Thr" or simply "T ”), cysteine (hereinafter referred to as “Cys” or simply “C”), tyrosine (hereinafter referred to as “Tyr” or simply “Y”), asparagine (hereinafter referred to as “Asn” or simply “N”), and glutamine (hereinafter referred to as "Gln" or simply "Q");

(3) Acidic amino acid group: aspartic acid (hereinafter referred to as "Asp" or simply "D") and glutamic acid (hereinafter referred to as "Glu" or simply "E");

(4) A group of basic amino acids: lysine (hereinafter referred to as "Lys" or simply "K"), arginine (hereinafter referred to as "Arg" or simply "R") and histidine (hereinafter referred to as "His" or simply "H" ).

In the present invention, the amino acids may be amino acids other than natural amino acids. Examples thereof may include selenocysteine, N-formylmethionine, pyrrolysine, pyroglutamic acid, cystine, hydroxyproline, hydroxylysine, thyroxine, O-phosphoserine, desmosine, β-alanine, sarcosine, ornithine, creatine, γ-aminobutyric acid, opine, theanine, tricholic acid , kainic acid, domoic acid, and acromelic acid, which occur naturally in peptides or proteins and may include: N-protected amino acids such as norleucine, Ac-amino acid, Boc-amino acid, Fmoc-amino acid, Trt-amino acid, and Z-amino acid ; C-protected amino acids such as tert-butyl ether, benzyl ether, cyclohexyl ether and amino acid fluorenyl ester; and other amino acids that do not occur naturally, including diamine, ω-amino acid, β-amino acid, γ-amino acid, Tic derivatives of amino acids and aminophosphonic acid. Amino acids other than the 20 "natural amino acids" are not limited thereto, and are collectively referred to as "non-natural amino acids" in the present invention for convenience.

[0029] 2-2. HTRA1 inhibitory peptide

The HTRA1 inhibitory peptide of the present invention is a SPINK2 mutant that at least partially retains the SPINK2 backbone (hereinafter referred to as "SPINK2 mutant"), and inhibits or represses the protease activity of HTRA1 or a fragment that retains its enzymatic activity (hereinafter referred to as "functional fragment") (such inhibition and suppression are collectively referred to as "HTRA1 inhibition activity").

[0030] The HTRA1 that is the target of the inhibitory peptide of the present invention is preferably mammalian HTRA1, more preferably primate HTRA1, and even more preferably human HTRA1. The amino acid sequence of full-length mature human HTRA1 (hereinafter referred to as "HTRA1 (full))" has the amino acid sequence shown in SEQ ID NO: 53 (Figure 65). This amino acid sequence consists of positions 23-480 and does not contain a signal sequence consisting of positions 1-22. The amino acid sequence of a functional fragment of human HTRA1 (hereinafter referred to as "HTRA1 (catalyte)") is not particularly limited, as long as the functional fragment retains protease activity. Examples thereof may include a functional fragment consisting of 158Gly-373Lys in SEQ ID NO: 53 (FIG. 65) and a functional fragment containing 158Gly-373Lys. HTRA1 or a functional fragment thereof which is the target of an inhibitory peptide of the present invention is also referred to as "HTRA1 protease". The HTRA1 protease is preferably derived from vertebrates, more preferably from mammals, even more preferably from primates, and most preferably from humans, and can be obtained by isolation from tissues or cells of any of these animals or by a method known to those skilled in the art such as recombination. genes, in vitro translation, or peptide synthesis. HTRA1 or a functional fragment thereof may be linked to a signal sequence, an immunoglobulin Fc region, a tag, a marker, or the like.

[0031] HTRA1 inhibition activity can be assessed using HTRA1 protease activity as an indicator. For example, HTRA1 or a functional fragment thereof, a substrate and an inhibitory peptide of the present invention or a candidate thereof are brought into contact with each other. In this case, when the protease activity of HTRA1 is 70% or less, 50% or less, 30% or less, 20% or less, 10% or less, 5% or less, 1% or less, or 0% compared to in the presence of a control or in the absence of an inhibitor or candidate thereof, then HTRA1 inhibition occurs with an inhibitory activity of 30% or more, 50% or more, 70% or more, 80% or more, 90% or more, 95% or more, 99% or higher or 100% respectively. The HTRA1 inhibition activity may vary depending on reaction conditions, substrate type, concentration, etc. Examples of reaction conditions may include, but are not limited to, the conditions described in the examples. Enzyme activity can be assessed by adding a peptide substrate or protein substrate to a certain concentration of HTRA1 and reacting the mixture for a certain period of time, followed by fluorescence detection of the peptide substrate or detection of the protein substrate using SDS-PAGE, Western blot, liquid chromatography, or the like. For example, phosphate buffered saline (hereinafter referred to as "PBS"), borate buffer (50 mM borate, pH 7-9, e.g. pH 8.5), or Tris buffer (50 mM Tris, pH 6-9, e.g., pH 8.0) can be used as a buffer solution. NaCl (50 to 300 mM, eg 150 mM) or a detergent such as CHAPS or octyl-D-glucopyranoside may be added to the reaction system, although the addition is not limited thereto.

[0032] Examples of a substrate for the HTRA1 protease include, but are not limited to, endogenous substrates, exogenous substrates, and synthetic substrates. Examples of a human endogenous substrate include vitronectin. Examples of synthetic substrate include, without limitation, H2-Opt (Mca-IRRVSYSFK(Dnp)K), β-casein, and other HTRA1 substrates. The HTRA1 inhibitory activity (IC ₅₀ or Ki) of the peptide of the present invention is 1 μM or less, more preferably 100 nM or less.

[0033] Preferably, the inhibitory peptide of the present invention does not inhibit or suppress the activity of a protease other than HTRA1, or has a relatively weak degree of inhibition or suppression of such activity. In other words, the inhibitory peptide of the present invention preferably has high specificity for HTRA1. Preferably, the inhibitory peptide of the present invention does not inhibit or suppress the activity of proteases such as trypsin, α-chymotrypsin, tryptase, plasmin, thrombin, matriptase, protein C, tissue plasminogen activator (tPA), urokinase (uPA), plasmin, and plasma kallikrein, or has a relatively weak degree of inhibition or suppression of such activities. Such a preferred peptide of the present invention does not show an adverse reaction caused by the inhibition or suppression of the activity of other proteases, and it can be preferably used as a therapeutic agent or prophylactic agent for a disease associated with HTRA1 (listed below).

As mentioned above, HTRA1, which is the target of the peptide of the present invention, preferably comes from vertebrates, more preferably from mammals, even more preferably from primates, and most preferably from humans. Alternatively, the HTRA1 that is the target of the peptide of the present invention may be derived from a non-human animal, such as a rodent such as a rat or mouse, or a primate such as a cynomolgus monkey, marmoset, or rhesus monkey. A peptide having an HTRA1 inhibitory activity derived from a non-human animal can be used for diagnosis, examination, treatment or prevention, etc. HTRA1 related disease in a non-human animal. When such a peptide also inhibits human HTRA1, a non-human animal can be used: in preclinical studies and in the development of a peptide for use as a therapeutic or prophylactic agent for a disease associated with human HTRA1; or to conduct a pharmacological study or a pharmacokinetic study using a non-human animal as an animal model of the disease; or to perform a safety test, a toxicity test, or the like. using a non-human animal as a healthy animal.

[0034] The HTRA1 inhibitory peptide of the present invention has the following advantages: lower molecular weight than other biological molecules, such as antibodies, which are used as drugs and diagnostics in the field; relatively easy to obtain (described below); excellent physical properties in terms of tissue penetration, storage stability, thermal stability and the like; and a wide range of choice of route of administration, mode of administration, preparation, and the like for use as a pharmaceutical composition (listed below). The blood half-life of the peptide of the present invention used as a pharmaceutical composition can be adjusted to be prolonged by applying a known method such as adding a biological molecule or polymer, and thus increasing the molecular weight of the peptide. The molecular weight of such an HTRA1 inhibitory peptide of the present invention is below 10,000, preferably below 8,000, and more preferably between about 7,000 and 7,200. from 15Cys-63Cys (hereinafter referred to as "fragment containing six Cys residues") and having HTRA1 inhibitory activity is also included in the term "HTRA1 inhibitory peptide" of the present invention. The molecular weight of the variable loop fragment is below 2500, and preferably about 1800-2000. The molecular weight of the fragment containing six Cys residues is below 6000, preferably about 5300-5500.

[0035] A SPINK2 mutant that at least partially retains the SPINK2 backbone (hereinafter referred to as "SPINK2 mutant") included within the scope of the term "HTRA1 inhibitory peptide" of the present invention can bind to HTRA1 and preferably is able to bind to HTRA1 mammals, more preferably primate HTRA1 and even more preferably human HTRA1. Such a peptide that binds to HTRA1 recognizes or binds to a partial peptide, incomplete conformation, or the like of HTRA1 (such recognition and binding effects are collectively referred to as "target binding activity") hereinafter.

In one aspect, the inhibitory peptide of the present invention is capable of binding to an immunogenic fragment of HTRA1. An immunogenic fragment of HTRA1 has one, two or more epitopes, mimotopes, or other antigenic determinants and is therefore capable of inducing an immune response or capable of eliciting an antibody against the fragment.

Binding of the SPINK2 mutant of the present invention to HTRA1 or an immunogenic fragment thereof can be assessed, measured, or determined using a method known to those skilled in the art, such as detectable binding affinity (ELISA, surface plasmon resonance (hereinafter referred to as "SPR") (also called "BIAcore" method), isothermal titration calorimetry (hereinafter referred to as "ITC"), flow cytometry, immunoprecipitation method, etc.).

[0036] Examples of ELISA include a method that includes detection of an HTRA1 inhibitory peptide that recognizes and binds to HTRA1 immobilized on a plate. Biotin-streptavidin can be used to immobilize HTRA1, as well as, for example, an immobilized antibody that recognizes a label fused to HTRA1. Labeled streptavidin can be used to detect the HTRA1 inhibitory peptide, as well as, for example, a labeled detection antibody that recognizes a label fused to the HTRA1 inhibitory peptide. Labeling can use biotin as well as a method suitable for biochemical analysis such as HRP, alkaline phosphatase or FITC. For detection using enzymatic labeling, a chromogenic substrate such as TMB (3,3',5,5'-tetramethylbenzidine), BCIP (5-bromo-4-chloro-3-indolylphosphate), p-NPP (p-nitrophenyl phosphate) can be used. ), OPD (o-phenylenediamine), ABTS (3-ethylbenzothiazoline-6-sulfonic acid), or SuperSignal ELISA Pico chemiluminescent substrate (Thermo Fisher Scientific Inc.), fluorescent substrate such as QuantaBlu(TM) peroxidase fluorogenic substrate (Thermo Fisher Scientific Inc.) and a chemiluminescent substrate. When measuring the detection signal, an absorbance measurement plate reader, a fluorescence measurement plate reader, a luminescence measurement plate reader, an RI liquid scintillation counter, or the like can be used.

[0037] Examples of an instrument for use in SPR analysis may include BIAcore™ (GE Healthcare), ProteOn™ (Bio-Rad Laboratories, Inc.), SPR-Navi™ (Oy BioNavis Ltd.), Spreeta(TM) (Texas Instruments Inc. .), SPRi-PlexII(TM) (HORIBA, Ltd.) and Autolab SPR(TM) (Metrohm AG). Examples of an instrument for BLI application may include Octet(TM) (Pall Corp.).

[0038] Examples of an immunoprecipitation-based method include a method that includes detection of HTRA1 that recognizes and binds to an HTRA1 inhibitory peptide immobilized on beads. As beads, magnetic beads, agarose beads or the like can be used. Biotin-streptavidin can be used to immobilize the HTRA1 inhibitory peptide, as well as an antibody that recognizes the peptide or tag fused to the peptide, protein A or protein G, etc. The beads are separated by a magnet, centrifugation, or the like, and HTRA1 precipitated with the beads is detected using SDS-PAGE or Western blotting. Labeled streptavidin can be used to detect HTRA1, as well as, for example, a labeled detection antibody that recognizes a label fused to HTRA1. Labeling can use biotin, as well as a method suitable for biochemical analysis such as HRP, alkaline phosphatase or FITC. For detection using enzymatic labeling, you can use the same substrate as in ELISA. ChemiDoc™ (Bio-Rad Laboratories, Inc.), LuminoGraph (ATTO Corp.), or the like can be used to measure the detection signal.

[0039] In the present invention, the term "specific recognition", i.e. "specific binding" is used to refer to binding that is not non-specific adsorption. Examples of a criterion for determining whether a binding is specific may include the EC ₅₀ value in the binding activity in the ELISA. Other examples of the determination criterion may include the dissociation constant (hereinafter referred to as "K _D "). The K _D value of the HTRA1 inhibitory peptide of the present invention for HTRA1 is 1×10 ^-4 M or less, 1×10 ^-5 M or less, 5×10 ^-6 M or less, 2×10 ^-6 M or less, or 1 ×10 ^-6 M or less, more preferably 5×10 ^-7 M or less, 2×10 ^-7 M or less, or 1×10 ^-7 M or less, even more preferably 5×10 ^-8 M or less, 2×10 ^-8 M or less, or 1×10 ^-8 M or less, even more preferably 5×10 ^-9 M or less, 2×10 ^-9 M or less, or 1×10 ^-9 M or less . Other examples of determination criteria may include the results of an immunoprecipitation assay. The preferred HTRA1 inhibitory peptide of the present invention is immobilized on the beads and HTRA1 is added to them. The beads are then separated and HTRA1 precipitated with the beads is detected. In this case, the HTRA1 signal is detected.

Despite the above description, the ability to bind to HTRA1 or an immunogenic fragment thereof is not essential for the SPINK2 mutant serving as an inhibitory peptide of the present invention, provided that the SPINK2 mutant has HTRA1 inhibitory activity.

[0040] The inhibitory peptide of the present invention may be competitive in binding a protease substrate to HTRA1.

[0041] In some preferred aspects, the inhibitory peptide of the present invention has a retinal protective effect. For example, a preferred inhibitor of the present invention can suppress light-induced nuclear depletion in the outer nuclear layer in a model of light-induced retinal injury, as detailed in the examples. More HTRA1 protein was found in the vitreous in the light exposed group in this model compared to the no exposure group. Thus, the present invention discloses that: HTRA1 is involved in retinal damage; and HTRA1 inhibitory activity provides a protective effect on the retina.

[0042] The SPINK2 mutant serving as an inhibitory peptide of the present invention may have the activities, properties, functions, features, etc. described above, while its full-length amino acid sequence has a high sequence identity with the amino acid sequence of human SPINK2 wild type. The SPINK2 mutant of the present invention has 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more high sequence identity with the amino acid sequence (SEQ ID NO: 1: Figure 13) of human SPINK2.

[0043] The term "identity" is used to refer to a property that indicates the degree of similarity or relationship between two sequences. The identity (%) of amino acid sequences is calculated by dividing the number of identical amino acids or amino acid residues by the total number of amino acids or amino acid residues and multiplying the resulting numerical value by 100.

[0044] The term "gap" is used to denote a space in the alignment of two or more sequences as a result of a deletion and/or addition in at least one of the two or more sequences.

[0045] The identity between two amino acid sequences having completely identical amino acid sequences is 100%. Provided that one of the amino acid sequences has a substitution, deletion or addition of one, two or more amino acids or amino acid residues compared to the other amino acid sequence, the identity between the two amino acid sequences is lower than 100%. Examples of an algorithm or program for determining the identity of two sequences based on gaps may include those known to those skilled in the art such as BLAST (Altschul et al., Nucleic Acids Res., Vol. 25, p. 3389-3402, 1997), BLAST2 (Altschul et al., J. Mol. Biol., Vol. 215, p. 403-410, 1990) and Smith-Waterman (Smith et al., J. Mol. Biol., Vol. 147, p. 195 -197, 1981).

[0046] In the present invention, the term "mutated" is used to mean that one, two or more nucleotides or nucleotide residues or amino acids or amino acid residues are substituted, deleted or inserted into a nucleotide sequence or amino acid sequence in comparison with the naturally occurring sequence of a nucleic molecule acid or peptide. The amino acid sequence of the SPINK2 mutant of the present invention has one, two or more mutated amino acids or amino acid residues compared to the human SPINK2 amino acid sequence.

[0047] In an aspect of the present invention, the amino acid sequence of the SPINK2 mutant may be any of:

substitution of 1, 2, 3, 4, 5, 6 or 7 amino acids from 16Ser-22Gly with other amino acids or amino acid residues;

substitutions of 1, 2, 3, 4 or 5 amino acids from 24Pro-28Asn with other amino acids or amino acid residues;

replacing 1 or 2 amino acids from 39Ala-43Thr with other amino acids or amino acid residues in the amino acid sequence (SEQ ID NO: 1: FIG. 13) of human SPINK2;

or may have these amino acids as wild-type amino acids (2 amino acids or 2 amino acid residues included in the α-helix), provided that the tertiary structure of the backbone of the loop, consisting of amino acid residues at positions 16 to 30, etc., is capable of at least partially exhibit HTRA1 inhibitory activity; each of 15Cys, 23Cys, 31Cys, 42Cys, 45Cys and 63Cys preferably represents Cys, as in the wild type, to preserve natural disulfide bonds. 1, 2, 3, 4, 5, or 6 of these may be replaced with other amino acids to remove natural disulfide bonds or form an unnatural disulfide bond. Some preferred HTRA1 inhibitory peptides among the SPINK2 mutants of the present invention retain Cys at these 6 positions, as occurs in native SPINK2, and retain disulfide bonds. In some more preferred forms of such an inhibitory peptide, 15Cys-45Cys, 23Cys-42Cys and 31Cys-63Cys respectively form disulfide bonds.

[0048] When the amino acid sequence of such a SPINK2 mutant is contained in an HTRA1 inhibitory peptide, it is preferable that the three-dimensional structure consists of, for example, a loop structure consisting of 16Ser-30Val, a β-sheet consisting of a "β-chain" (1) , consisting of 31Cys and 32Gly, and β-chain (2), consisting of 57Ile-59Arg, α-helix, consisting of 41Glu-51Gly, or a loop structure, β-sheet or α-helix, similar to them or at least at least partially corresponding to them (or these positions) contained in the wild-type SPINK2 amino acid sequence should be retained to the extent that HTRA1 inhibition activity can be exhibited.

[0049] The amino acid sequences of some of the HTRA1 inhibitory peptides among the SPINK2 mutants of the present invention will be mentioned below. As indicated above, in the present invention, the term "amino acid residue" is also simply referred to as "amino acid".

[0050] Each of the first to eleventh Xaa (which are the same as X ₁ -X ₁₁ respectively) in the amino acid sequence shown in SEQ ID NO: 30 (FIG. 42) represents any arbitrary amino acid without particular limitation, provided that the resulting mutant binds to HTRA1 and inhibits HTRA1 activity. Next, the preferred amino acids X1-X11 will be described. However, these amino acids may include natural amino acids, ie. amino acids identical to the amino acids in the human wild type SPINK2 amino acid sequence.

[0051] X1 at position 1 is preferably Asp, Glu, Gly, Ser, or Ile, more preferably Asp or Gly, and even more preferably Gly;

X2 at position 16 is preferably Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Gln, Arg, Ser, Thr or Tyr, more preferably Ala, Asp, Gly, His, Lys, Leu, Met, Gln, Arg, Ser or Thr, even more preferably Ala, Gly, Lys, Leu, Ser or Thr, even more preferably Ala, Gly, Leu, Ser or Thr and even more preferably Ala or Ser;

X3 at position 17 is preferably Ala, Asp, Glu, Gly, His, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr or Tyr, more preferably Asp, Gly, His, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr or Tyr, even more preferably Asp, His, Lys, Met or Gln and even more preferably Asp or Gln;

X4 at position 18 is preferably Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Lys, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr, more preferably Asp, Phe, His, Met, Asn, Gln, Ser or Tyr, even more preferably Asp, Phe, His, Ser or Tyr, and even more preferably Phe or His;

X5 at position 19 is preferably Ala, Asp, Glu, Gly, His, Ile, Lys, Met, Asn, Gln, Arg, Ser, Thr, Val or Tyr, more preferably Ala, Asp, Glu, Gly, His, Lys, Met, Asn, Gln, Arg, Ser or Val, even more preferably Ala, Asp, Glu, Met or Asn and even more preferably Ala, Asp or Glu;

X6 at position 21 is preferably Ala, Glu, Phe, Gly, Ile, Leu, Met, Gln, Arg, Ser, Trp or Tyr, more preferably Glu, Phe, Ile, Leu, Met, Gln, Arg or Trp, even more preferably Met or Trp, even more preferably Met;

X7 at position 24 is preferably Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr, more preferably Asp, Glu, His, Pro, Gln, Ser, Thr, Val, Trp or Tyr, even more preferably Gln, Trp, Tyr or Val and even more preferably Tyr or Val;

X8 at position 26 is preferably Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Val, Tyr, more preferably Ala, Phe, His, Ile, Leu, Met, Gln, Arg, Ser, Val or Tyr, even more preferably Phe, Leu or Tyr and even more preferably Phe or Leu;

X9 at position 27 is preferably Glu, Phe, Leu, Ser, Thr or Tyr, more preferably Phe, Leu, Ser, Thr or Tyr, even more preferably Phe or Tyr, even more preferably Tyr;

X10 at position 39 is preferably Ala, Glu, Met or Val and more preferably Ala or Glu; and

X11 at position 43 is preferably Ala, Thr or Val, more preferably Thr or Val.

[0052] Wild-type amino acids X1-X11 are Asp, Ser, Gln, Tyr, Arg, Pro, Pro, His, Phe, Ala, and Thr, respectively. Position 20 represents Leu, position 22 represents Gly, position 25 represents Arg, and position 28 represents Asn.

[0053] In the present invention, one, more, or more amino acids may be additionally added to the N-terminal portion of the first amino acid. Examples of such amino acids that can be added include Ser-Leu and an amino acid sequence consisting of an S-tag and a linker (SEQ ID NO: 31: Fig. 43).

[0054] One or more amino acids may be additionally added to the 63Cys located at the C-terminus. Examples of such an amino acid sequence may include an amino acid sequence having 64Gly at the C-terminus and an amino acid sequence having 65Gly at the C-terminus by adding Gly-Gly. Examples of such an amino acid that can be added include Gly-Gly and the C-terminal hexamer (SEQ ID NO: 32: Figure 44).

[0055] A more preferred form of the amino acid sequence obtained by adding other amino acids to the N-terminus and/or C-terminus of the amino acid sequence shown in SEQ ID NO: 30 (Figure 42) or the amino acid sequence obtained from SEQ ID NO: 30, includes the amino acid sequence shown in any of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23-29 (Fig.15, 17, 19, 21 23, 25, 27 , 29, 31, 33 and 35-41) and the amino acid sequence encoded by the nucleotide sequence shown in any of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22 (Fig. 16 , 18, 20, 22, 24, 26, 28, 30, 32 and 34). An even more preferred form thereof comprises the amino acid sequence shown in any of SEQ ID NOs: 24, 28 and 29 (FIGS. 36, 40 and 41).

[0056] In the present invention, a peptide obtained by substitution, addition and/or deletion of one, two or more amino acids to or from a mutant SPINK2 peptide, or an N-terminal and/or C-terminal adduct of a mutant SPINK2 peptide (hereinafter referred to as "parent peptide" ) refers to "parent peptide derivative" or "parent peptide derivative" (eg, Example 6). Such a "derivative" is also within the scope of the term "peptide" of the present invention.

[0057] The amino acid sequence of the SPINK2 mutant included within the scope of the term "inhibitory peptide" of the present invention may contain a naturally occurring or mutated amino acid sequence, or an amino acid sequence in fragments other than X1-X11, i.e. at positions 2Pro to 15Cys, 20Pro, 22Gly, 23Cys, 25Arg, 28Asn-38Tyr, 41Glu, 42Cys and 44Thr-63Cys, in the amino acid sequence (SEQ ID NO: 1: FIG. 13) of wild-type human SPINK2. For example, a SPINK2 mutant may have a mutation at one, two, or more positions, provided that HTRA1 inhibitory activity or HTRA1 folding is not at least partially hindered or impaired. Such a mutation is achieved using a standard method known to those skilled in the art. Typical examples of a mutation in an amino acid sequence may include a substitution, deletion or addition of one, two or more amino acids. Examples of substitution may include conservative substitution. A conservative substitution results in the replacement of a particular amino acid residue with an amino acid residue that is chemically similar to it in terms of not only bulk but also polarity. Examples of conservative substitutions are described elsewhere in this specification. On the other hand, fragments other than X1-X11 may undergo a non-conservative substitution of one, two or more amino acids, provided that HTRA1 inhibitory activity or HTRA1 folding is not at least partially hindered or impaired.

[0058] In the amino acid sequence of the SPINK2 mutant serving as the inhibitory peptide of the present invention, X1-X11 preferably represent the amino acids X1-X11, respectively, in any of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23-29 (FIGS. 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 and 35-41) and more preferably SEQ ID NOs: 24, 28 and 29 (FIGS. 36, 40, and 41), and fragments other than X1-X11 may have an amino acid or amino acid sequence that does not at least partially interfere with or impair HTRA1 inhibitory activity or folding.

Examples of the amino acid sequence of the SPINK2 mutant serving as the HTRA1 inhibitory peptide of the present invention may include any of the following amino acid sequences (a) to (e):

(a) the amino acid sequence shown in any of the sequences of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23-29 (Fig. 15, 17, 19, 21, 23 , 25, 25, 29, 31, 33 and 35-41);

(b) an amino acid sequence encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence complementary to the nucleotide sequence encoding the amino acid sequence (a) and encodes an amino acid sequence contained in a peptide having an HTRA1 inhibitory activity;

(c) an amino acid sequence that is derived from the amino acid sequence of (a) by substitution, deletion, addition and/or insertion from 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2 or 1 amino acids, and is contained in a peptide having an HTRA1 inhibitory activity;

(d) an amino acid sequence that shares 60%, 70%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98% or 99% or more identity with the amino acid sequence of (a) and contained in a peptide having HTRA1 inhibitory activity; and

(e) the amino acid sequence encoded by the nucleotide sequence shown in any of the sequences of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22 (Fig. 16, 18, 20, 22, 24 , 26, 28, 30, 32 and 34).

[0059] However, the nucleic acid molecule encoding the HTRA1 inhibitory peptide is not limited to (a)-(e). Any nucleic acid molecule containing a nucleotide sequence encoding an amino acid sequence contained in a SPINK2 mutant having HTRA1 inhibition activity, and preferably the amino acid sequence shown in SEQ ID NO: 30 (Fig. 42), is included within the scope of the term "nucleic acid molecule, encoding an HTRA1 inhibitory peptide."

[0060] A mutation can be introduced into the inhibitory peptide of the present invention to improve its folding stability, thermal stability, storage stability, blood half-life, water solubility, biological activity, pharmacological activity, secondary effect, etc. For example, a new reactive group such as Cys can be introduced into the peptide by mutation to conjugate the inhibitory peptide to an additional compound such as polyethylene glycol (PEG), hydroxyethyl starch (HES), biotin, peptide or protein. In the present invention, an HTRA1 inhibitory peptide may be added, linked, or linked to an additional molecule. Such conjugates are collectively referred to as "HTRA1 inhibitory peptide conjugate". In the present invention, the term "conjugate" is used to refer to a peptide molecule of the present invention, or a fragment thereof, added to, associated with or connected to an additional molecule. The term "conjugate" or "conjugation" includes the form in which a particular molecule is bonded or linked through an agent or the like suitable for linking the particular molecule to an amino acid side chain, including a chemical compound, such as a crosslinking agent, to the N-terminus and/or the C-terminus of the peptide of the present invention through a synthetic chemistry approach, a genetic engineering approach, or the like. Examples of such a "molecule" for prolonging blood half-life may include polyalkylene glycol molecules such as polyethylene glycol (PEG), hydroxyethyl starch, fatty acid molecules such as palmitic acid, immunoglobulin Fc regions, immunoglobulin CH3 domains, immunoglobulin CH4 domains, albumin or fragments thereof, albumin-binding peptides, albumin-binding proteins such as streptococcal G protein and transferrin. Alternatively, the "molecule" may be linked to the peptide of the present invention via a linker, such as a peptide linker.

[0061] The HTRA1 inhibitory peptide of the present invention can be conjugated with an additional drug to enhance or increase pharmacological activity. In the field of antibodies, a technique or form known to those skilled in the art, such as antibody-drug conjugates (ADCs), constitutes some aspects of the present invention by replacing an antibody with a peptide of the present invention.

[0062] The HTRA1 inhibitory peptide of the present invention may further comprise one, two or more molecules that exhibit binding affinity, inhibitory activity, antagonistic activity, agonist activity, or the like for a target molecule other than HTRA1, or it can be conjugated to such molecules. Examples of "molecules" may include antibodies or fragments thereof, and proteins having a backbone other than the antibody, such as SPINK2 mutants, or fragments thereof. In the field of antibodies, a technique or form known to those skilled in the art, such as multispecific antibodies and bispecific antibodies, constitutes certain aspects of a conjugate of the present invention by replacing at least one, two or more "antibodies" contained therein with a peptide according to the present invention.

[0063] The peptide of the present invention or its precursor may contain a signal sequence. A signal sequence present or added to the N-terminus of a particular polypeptide or precursor thereof is useful for delivering the polypeptide to a specific cell compartment, such as the periplasmic space of E. coli or the endoplasmic reticulum of a eukaryotic cell. Many signal sequences are known to those skilled in the art, and the signal sequence can be selected according to the host cells. Examples of a signal sequence for secretion of the desired peptide into the periplasmic space of E. coli may include OmpA. The form containing the signal sequence can also be included in some aspects of the "conjugate" of the present invention.

[0064] The peptide of the present invention can be labeled in advance, thereby allowing purification by affinity chromatography. The peptide of the present invention may include, for example, biotin, Strep(TM) tag, Strep II(TM) tag, oligohistidine such as His6, polyhistidine, immunoglobulin domain, maltose binding protein, glutathione S-transferase (GST) , a calmodulin-binding peptide (CBP), a hapten such as digoxigenin or dinitrophenol, an epitope tag such as FLAG(TM), a myc tag or an HA tag (hereinafter referred to collectively as an "affinity tag"), at its C-terminus . The labeled form may also be included in some aspects of the "conjugate" of the present invention. In general, the conjugate of the present invention may be a peptide (polypeptide).

[0065] The peptide of the present invention may contain a labeling molecule. In particular, the peptide of the present invention can be conjugated to a label molecule such as an enzyme label, a radioactive label, a color label, a fluorescent label, a dye label, a luminescent label, a hapten, digoxigenin, biotin, a metal complex, a metal, or colloidal gold. The form containing the labeling molecule can also be included in some aspects of the "conjugate" of the present invention.

[0066] The inhibitory peptide of the present invention may contain any natural amino acids and non-natural amino acids in its peptide fragment, and may contain L-amino acid and D-amino acid as a natural amino acid.

[0067] The amino acid sequence of the inhibitory peptide of the present invention may contain any natural amino acids and non-natural amino acids, and may contain L-amino acid and D-amino acid as a natural amino acid.

[0068] The inhibitory peptide of the present invention may be in the form of a monomer, dimer or trimer, or a higher order oligomer or multimer. The dimer or trimer or higher oligomer or multimer may be any of a homoform consisting of the same monomers and a heteroform consisting of two or more different monomers. The monomer can diffuse rapidly and penetrates excellently, for example, into tissues. The dimer, oligomer and multimer may have an advantageous aspect such as high local affinity or binding activity for the target molecule, low dissociation rate or high HTRA1 inhibitory activity. In addition to spontaneous dimerization, oligomerization, and multimerization, intentional dimerization, oligomerization, and multimerization are achieved by introducing a jun-fos domain, a leucine zipper, or the like into the inhibitory peptide of the present invention.

[0069] The inhibitory peptide of the present invention can bind to one, two or more target molecules or inhibit the activity of target molecules in the form of a monomer, dimer or trimer or more of a higher order oligomer or multimer.

[0070] Examples of the form that the inhibitory peptide of the present invention can take may include, but are not limited to, isolated forms (lyophilized preparations, solutions, etc.), conjugates as described above, and forms associated with other molecules (immobilized forms associated with another molecule, forms associated with a target molecule, etc.). The form compatible with expression, purification, use, storage, and the like may be arbitrarily selected.

[0071] 3. Identification of HTRA1 inhibitory peptide

The HTRA1 inhibitory peptide can be identified in a manner well known to those skilled in the art using starting material such as the amino acid sequence of SPINK2 or an amino acid sequence (e.g., an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23-29, or the group consisting of Fig. 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 and 35-41) HTRA1 α-inhibitory peptide of the present invention, a nucleotide sequence encoding an amino acid sequence, or a nucleic acid molecule containing such a nucleotide sequence. As a preferred example, an HTRA1 inhibitory peptide can be identified from a human SPINK2 mutant library using HTRA1 inhibitory activity as an index, which can be combined with HTRA1 binding activity as another index.

[0072] For example, a nucleic acid molecule serving as a starting material can be mutagenized and transfected into an appropriate bacterial host or eukaryotic host using a recombinant DNA technique. The SPINK2 mutant library is known as a method for identifying a binding agent or inhibitor of a target molecule. For example, the disclosure of WO2012/105616 is also incorporated herein by reference in its entirety. After expressing the mutagenized nucleotide sequence in an appropriate host, a clone in which a SPINK2 mutant having the desired properties, activity, function, etc. is associated with its genotype can be enriched and/or selected from a library and identified. The enrichment and/or selection of a clone uses a method known to those skilled in the art, such as the bacterial display method (Francisco J.A. et al. (1993) Proc. Natl. Acad. Sci. USA, Vol. 90, p. 10444-10448) , yeast display method (Boder E.T. et al. (1997) Nat. Biotechnol., Vol. 15, p. 553-557), mammalian cell display method (Ho M. et al. (2009) Methods Mol Biol., Vol. 525: p. 337-52), phage display method (Smith G.P. (1985) Science., Vol. 228, p. 1315-1317), ribosome display method (Mattheakis L.C. et al. (1994) Proc. Natl. Acad. Sci. USA, Vol. 91, No. 19, p. 9022-9029), a nucleic acid display method such as mRNA display (Nemoto N. et al. (1997) FEBS Lett., Vol. 414, No. 2, p. 405-408), or the colony screening method (Pini A. et al. (2002) Comb. Chem. High Throughput Screen., Vol. 5, p. 503-510). The nucleotide sequence of the SPINK2 mutant present in the clone thus selected and identified can be determined to establish the amino acid sequence encoded by the nucleotide sequence as the amino acid sequence of the SPINK2 mutant present in the clone, i. HTRA1 inhibitory peptide.

[0073] The SPINK2 mutant of the present invention can be obtained, for example, by mutagenization of native SPINK2. The term "mutagenesis" is used to mean that one, two or more amino acids at their respective positions in a particular amino acid sequence can be replaced with other amino acids or deleted, or an amino acid missing from the amino acid sequence can be added or inserted into it. Such a deletion or such an addition or insertion may change the length of the sequence. In the SPINK2 mutant of the present invention, mutagenesis may preferably take place at one, two or more positions X1 to X11 in the amino acid sequence shown in SEQ ID NO: 30 (FIG. 42).

[0074] However, a form that retains, after such preferential mutagenesis, the naturally occurring amino acids at one, two, or more positions X1 to X11, i. e. the same amino acid that is present at a particular position in the natural amino acid sequence is also included within the scope of the term "mutant" provided that at least one amino acid is mutated in general. Similarly, in an aspect of the present invention, a form which retains, after mutagenesis at one or more positions in the group other than X1-X11, the naturally occurring amino acids at those positions, i.e. the same amino acid that is present at a specific position in the natural amino acid sequence is also within the scope of the term "mutant" provided that at least one amino acid is mutated in general.

[0075] The term "random mutagenesis" is used to mean that, with respect to a certain position in the sequence, one, two or more different amino acids are introduced with a certain probability at a position by mutagenesis. The probabilities of introducing at least two different amino acids may not always be the same. The present invention does not exclude that said at least two different amino acids are included from a naturally occurring amino acid (as one of the amino acids). Such a case is also included in the scope of the term "random mutagenesis".

[0076] You can use the standard method known to experts in this field, as a method of random mutagenesis at a specific position. Mutagenesis can be achieved, for example, by PCR (polymerase chain reaction) using a mixture of synthetic oligonucleotides comprising a composition of degenerate nucleotides at a specific position in the sequence. For example, use of the codon NNK or NNS (N = adenine, guanine, cytosine, or thymine; K = guanine or thymine; and S = guanine or cytosine) causes mutagenesis with the introduction of any of all 20 natural amino acids, as well as a stop codon, while while the use of the VVS codon (V = adenine, guanine or cytosine) excludes the possibility of introducing Cys, Ile, Leu, Met, Phe, Trp, Tyr and Val and causes mutagenesis with the introduction of any of the remaining 12 natural amino acids. For example, the use of the NMS codon (M = adenine or cytosine) excludes the possibility of introducing Arg, Cys, Gly, Ile, Leu, Met, Phe, Trp and Val and causes mutagenesis with the introduction of any of the remaining 11 natural amino acids. A specific codon, an artificial codon, or the like can be used for mutagenesis with the introduction of a non-natural amino acid.

[0077] Site-directed mutagenesis can also be performed by using structural information about the target, containing the conformation and/or peptide against the target or the wild-type peptide from which the peptide is derived. In the present invention, a site-directed mutation can be introduced by using structural information, including higher order information about the target HTRA1 and/or SPINK2 mutant to the target or wild-type SPINK2, or a complex between them. In one example, a SPINK2 mutant having HTRA1 inhibitory activity is identified. A crystalline complex of HTRA1 and the SPINK2 mutant is then obtained and subjected to X-ray crystallography. Based on the results of the analysis, the site on the HTRA1 molecule to which the SPINK2 mutant binds and the amino acid residue in the SPINK2 mutant involved in the interaction with the site is identified. Structural information obtained by such procedures can be correlated with HTRA1 inhibitory activity. Based on such a structure-activity correlation, substitution of an amino acid at a specific position by a specific amino acid, insertion or deletion of an amino acid at a specific position, or the like can be designed to actually confirm the activity of HTRA1.

[0078] Mutagenesis can also be achieved using, for example, a nucleotide moiety with modified base pair specificity, such as inosine.

[0079] In addition, random position mutagenesis is achieved, for example, by error-prone PCR using a DNA polymerase such as Taq DNA polymerase, which lacks an error correction function and produces a high error rate, or chemical mutagenesis.

[0080] The HTRA1 inhibitory peptide can be enriched and/or selected from a library, such as a phage library or a colony library, that is suitable for the appropriate screening method and is known to those skilled in the art using bacterial display, yeast display, mammalian cell display , phage display, ribosomal display, nucleic acid display, colony screening or the like. These libraries can be constructed using a vector and method, such as a phagemid for a phage library or a cosmid for colony screening, which are suitable for each library and known to those skilled in the art. Such a vector may be a virus or a viral vector that infects prokaryotic cells or eukaryotic cells. Such recombinant vectors can be obtained by a method known to those skilled in the art, such as genetic manipulation.

[0081] Bacterial display is a method of fusing a desired protein, for example, to an E. coli outer membrane lipoprotein (Lpp) region and an outer membrane protein, OmpA, and displaying the desired protein on the E. coli surface. A group of DNA resulting from random mutagenesis of a nucleotide sequence encoding the amino acid sequence of a particular protein is inserted into vectors suitable for bacterial display, and bacterial cells can be transformed with the vectors to obtain a library displaying a group of randomly mutagenized proteins on the surface of the transformed bacterial cell (Francisco J.A. et al. (1993) Proc. Natl. Acad. Sci. USA, Vol. 90, pp. 10444-10448).

[0082] Yeast display is a method of fusing a protein of interest to a coat protein such as α-agglutinin, which is found on the yeast cell surface, and displaying the protein of interest on the surface of the yeast. α-agglutinin contains a C-terminal hydrophobic region with a putative glycosylphosphatidylinositol anchor (GPI) attachment signal, a signal sequence, an active domain, a cell wall domain, and the like. The desired protein can be displayed on the yeast cell surface by manipulation. A group of DNA resulting from random mutagenesis of a nucleotide sequence encoding the amino acid sequence of a particular protein is inserted into vectors suitable for yeast display, and yeast cells can be transformed with the vectors to obtain a library displaying a group of randomly mutagenized proteins on the cell surface of transformed yeast (Ueda M. & Tanaka A., Biotechnol. Adv., Vol. 18, p. 121-, 2000; Ueda M. & Tanaka A., J. Biosci. Bioeng., Vol. 90, p. 125-, 2000, etc. d.).

[0083] Animal cell display is a method of fusing the desired protein, for example, to the transmembrane region of a membrane protein typified by platelet growth factor receptor (PDGFR), and displaying the desired protein on the surface of mammalian cells (e.g., HEK293) and Chinese ovary cells. hamster (CHO). A group of DNA resulting from random mutagenesis of a nucleotide sequence encoding the amino acid sequence of a particular protein is inserted into vectors suitable for display on animal cells, and animal cells can be transfected with the vectors to obtain a library displaying a group of randomly mutagenized proteins on the surface of transfected animal cells (Ho M. et al (2009) Methods Mol Biol, Vol 525: pp 337-52).

[0084] The desired library, displayed on cells, such as yeast cells, bacterial cells, or animal cells, can be incubated in the presence of the target molecule or subjected to contact with the target molecule. For example, cells including the library are incubated for a certain time with HTRA1 modified with biotin or the like. A support, such as magnetic beads, is then added to them, and the cells are separated from the support. The support can then be washed to remove non-specific adsorbed matter and bound substance to recover a group of cells that exhibit a peptide, a collection of peptides, or an enriched collection of peptides associated with the support (to which HTRA1 is bound). Similarly, a group of cells displaying a peptide, a collection of peptides, or an enriched collection of peptides associated with a support (to which HTRA1 is bound), or a group of cells displaying a peptide, a collection of peptides, or an enriched collection of collections associated with HTRA1, can be isolated using magnetically activated cell sorting (MACS) after adding magnetic beads or using FACS after staining cells with anti-HTRA1 antibody, respectively. For example, it is possible to block (saturate) a non-specific adsorption site and/or a binding site. It is possible to include a blocking step here, provided that the blocking is done in an appropriate way. The expression peptide, peptide collection, or enriched peptide collection vector thus obtained is isolated, and the nucleotide sequence of the polynucleotide inserted into the vector can be determined to determine the amino acid sequence encoded by the nucleotide sequence. In addition, host cells can be re-transfected with the vector, and the cycle of operations above can be repeated one to several times to greatly enrich the collection of peptides that bind to the target molecule.

[0085] For example, in the case of phage display, the phagemid is a bacterial plasmid containing a plasmid origin of replication as well as a second origin of replication derived from a single-stranded bacteriophage. A cell having a phagemid can replicate the phagemid by single-stranded replication by co-infection with M13 or a similar helper bacteriophage. In particular, single-stranded phagemid DNA is packaged in an infectious particle coated with a bacteriophage coat protein. Thus, phagemid DNA can be formed as a clone of double-stranded plasmid DNA in an infected bacterium, while phagemid can be formed as a bacteriophage-like particle from the culture supernatant of co-infected cells. The bacteriophage-like particle is introduced into a bacterium having an F-pilus to infect the bacterium with DNA, so that the particle itself can be converted into a plasmid.

[0086] A hybrid gene containing a polynucleotide having a nucleotide sequence encoding the amino acid sequence of a test peptide and a bacteriophage coat protein gene is inserted into a phagemid, and the bacterium is infected with the resulting phagemid. The cells are cultured such that the peptide can be expressed or displayed on a bacterium or a phage-like particle, or can be produced as a fusion protein with an envelope protein in a phage particle or in a bacterium culture supernatant.

[0087] For example, a fusion gene containing a polynucleotide and a bacteriophage gpIII coat protein gene is inserted into a phagemid, and E. coli is co-infected with the resulting phagemid and M13 or a helper phage similar to it, so that the fusion protein containing the peptide and coat protein, can be produced and released into the culture supernatant of E. coli.

[0088] In the case of using various cyclic or non-cyclic vectors, for example, a viral vector instead of a phagemid, a peptide having an amino acid sequence encoded by the nucleotide sequence of a polynucleotide inserted into the vector may be expressed or displayed on cells or virus-like particles carrying the vector, or may be produced and released into the cell culture supernatant according to a method known to those skilled in the art.

[0089] The peptide expression library thus obtained can be incubated in the presence of the target molecule or subjected to contact with the target molecule. For example, an HTRA1-immobilized support is incubated for a certain period of time with a mobile phase containing the library. The mobile phase is then separated from the support. The support is then washed to remove non-specific adsorbed matter and bound matter. The peptide, peptide collection, or enriched collection of peptides associated with the support (to which HTRA1 is bound) can be recovered by elution. Elution may be performed non-selectively, for example, in the presence of relatively high ionic strength, low pH, moderate denaturation conditions, or a chaotropic salt, or may be performed selectively by adding a soluble target molecule such as HTRA1, an antibody that binds to the target molecule, naturally occurring ligand, substrate or the like and competition with the immobilized target molecule. For example, a non-specific adsorption site and/or a binding site can be blocked. The blocking stage can be included here if the blocking is performed in an appropriate way.

[0090] The expression peptide, peptide collection, or enriched peptide collection vector thus obtained is isolated, and the nucleotide sequence of the polynucleotide inserted into the vector can be determined to establish the amino acid sequence encoded by the nucleotide sequence. In addition, host cells can be transfected again with the vector, and the cycle of operations mentioned above can be repeated one to several times to significantly enrich the collection of peptides that bind to the target molecule.

[0091] Ribosomal display is a method that uses, for example, an mRNA that encodes a desired protein and does not have a stop codon, and a cell-free protein synthesis system, and thereby provides in vitro synthesis of a desired protein molecule corresponding to its mRNA, and a ribosome connected to each other. A library displaying a group of randomly mutagenized proteins on ribosomes can be obtained by using an mRNA group obtained by random mutagenesis of a nucleotide sequence encoding the amino acid sequence of a certain protein and a cell-free protein synthesis system (Mattheakis L.C. et al. (1994) Proc. Natl. Acad. Sci (USA, Vol. 91, No. 19, pp. 9022-9029).

[0092] Nucleic acid display, also referred to as mRNA display, is a technique that uses, for example, a linker, such as puromycin, structurally similar to the 3' end of tyrosyl-tRNA, and thereby allows the synthesis of a desired protein molecule encoding its mRNA and ribosome linked to each other. This method uses a cell-free protein synthesis system rather than living cells, and therefore provides in vitro synthesis. A library displaying a group of randomly mutagenized proteins on a ribosome can be obtained by using an mRNA group obtained by random mutagenesis of a nucleotide sequence encoding the amino acid sequence of a particular protein, a linker such as puromycin, and a cell-free protein synthesis system (Nemoto N. et al. ( 1997) FEBS Lett., Vol. 414, No. 2, pp. 405-408).

[0093] A peptide display library prepared using a cell-free synthesis system, such as a ribosome display or nucleic acid display, can be incubated in the presence of a target molecule or contacted with a target molecule. For example, an HTRA1-immobilized support is incubated for a certain period of time with a mobile phase containing the library. The mobile phase is then separated from the support. The support is then washed to remove non-specific adsorbed matter and bound matter. The peptide, collection of peptides, or enriched collection of peptides associated with the support (to which HTRA1 is bound) can be isolated by elution. Elution may be performed non-selectively, for example, in the presence of relatively high ionic strength, low pH, moderate denaturation conditions, or a chaotropic salt, or may be performed selectively by adding a soluble target molecule such as HTRA1, an antibody that binds to the target molecule, naturally occurring ligand, substrate or the like and competition with the immobilized target molecule. For example, a non-specific adsorption site and/or a binding site can be blocked. The blocking stage can be included here if the blocking is performed by the appropriate method.

[0094] The expressed peptide nucleic acid, peptide collection, or enriched peptide collection thus obtained is isolated. In the case of mRNA, the nucleotide sequence can be determined after a reverse transcription reaction in cDNA to establish the amino acid sequence encoded by the nucleotide sequence. In addition, the isolated nucleic acid is transcribed into mRNA, and the cycle of operations above can be repeated one to several times to significantly enrich the collection of peptides that bind to the target molecule.

[0095] If a peptide, peptide collection, or enriched peptide collection is pre-conjugated with an affinity tag, then the peptide or collection can be effectively purified. For example, a collection of peptides is pre-conjugated to a labeled protease substrate such that the peptide can be eluted by cleavage via protease activity.

[0096] Based on the information obtained about the sequence and function of the peptide, etc., the obtained clone or library can be further mutagenized, and a peptide with improved functions (for example, HTRA1 inhibition activity), physical properties (thermostability, stability) can be obtained from the mutated library. during storage, etc.), in vivo kinetics (distribution and half-life in the blood), etc.

[0097] An HTRA1 inhibitory peptide can be identified by determining whether the resulting peptide has HTRA1 inhibitory activity.

[0098] The HTRA1 inhibitory peptide is preferably capable of maintaining a three-dimensional structure consisting of, for example, a loop structure consisting of 16Ser-30Val, a β-sheet consisting of a β-chain (1) consisting of 31Cys and 32Gly, and a β-chain (2) consisting of 57Ile-59Arg and an α-helix consisting of 41Glu-51Gly, or a loop structure, β-sheet, or α-helix similar to or at least partially corresponding to them (or these positions) contained in the amino acid sequence of the wild-type SPINK2, to the extent that HTRA1 inhibition activity can be exhibited. A more preferred HTRA1 inhibitory peptide can be identified using such a three-dimensional structure (full structure or incomplete structure) as part of the index.

[0099] 4. A nucleic acid molecule encoding an HTRA1 inhibitory peptide, a vector containing the nucleic acid molecule, a cell containing the nucleic acid molecule or a vector, and a method for producing a recombinant HTRA1 inhibitory peptide

The present invention also relates to a polynucleotide comprising a nucleotide sequence encoding an amino acid sequence contained in an HTRA1 inhibitory peptide (hereinafter referred to as "a nucleic acid molecule encoding an HTRA1 inhibitory peptide"), a recombinant vector having a gene insert, a cell carrying the gene, or a vector (hereinafter referred to as "a cell containing a nucleic acid molecule encoding an HTRA1 inhibitory peptide"), or a cell producing an HTRA1 inhibitory peptide (hereinafter referred to as an "HTRA1 inhibitory peptide producing cell").

[0100] Some preferred examples of a nucleic acid molecule encoding an HTRA1 inhibitory peptide of the present invention may include a nucleic acid molecule comprising any of the following nucleotide sequences (a) to (e) (hereinafter referred to as "HTRA1 inhibitory peptide nucleotide sequence" ), a nucleic acid molecule consisting of a nucleotide sequence containing an antibody gene sequence, and a nucleic acid molecule consisting of an antibody gene sequence:

(a) a nucleotide sequence encoding the amino acid sequence shown in any of the sequences of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23-29 (Fig. 15, 17, 19 , 21, 23, 25, 25, 29, 31, 33 and 35-41);

(b) the nucleotide sequence shown in any of the sequences of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22 (Fig. 16, 18, 20, 22, 24, 26, 28 , 30, 32 and 34);

(c) a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence complementary to the nucleotide sequence of (a) or (b) and encodes an amino acid sequence contained in a peptide having an HTRA1 inhibitory activity;

(d) a nucleotide sequence that is derived from the nucleotide sequence of (a) or (b) by substitution, deletion, addition and/or insertion from 1 to 20, from 1 to 15, from 1 to 10, from 1 to 8, from 1 to 6, 1-5, 1-4, 1-3, 1 or 2, or 1 nucleotide or nucleotide residue, and encodes an amino acid sequence contained in a peptide having an HTRA1 inhibitory activity; and

(e) a nucleotide sequence that shares 60%, 70%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98% or 99% or more identity with the nucleotide sequence of (a) or (b) and encodes an amino acid sequence contained in a peptide having an HTRA1 inhibitory activity.

[0101] However, the nucleic acid molecule encoding the HTRA1 inhibitory peptide is not limited to the nucleotide sequences (a) to (e). Any nucleic acid molecule containing a nucleotide sequence encoding an amino acid sequence contained in a SPINK2 mutant having HTRA1 inhibition activity, and preferably the amino acid sequence shown in SEQ ID NO: 30 (Fig. 42), is included within the scope of the term "nucleic acid molecule, encoding an HTRA1 inhibitory peptide."

[0102] One, two or more codons corresponding to each amino acid can be used to construct a nucleotide sequence encoding an amino acid sequence. Therefore, the nucleotide sequence encoding one amino acid sequence of a particular peptide can have many variations. To select such codons, the codons can be appropriately selected based on the codon usage of the host cells to be expressed to include the polynucleotide containing the nucleotide sequence or the vector containing the polynucleotide, or the frequency or rate of the plurality of codons used can be adjusted accordingly. For example, in the case of using Escherichia coli cells as host cells, the nucleotide sequence can be constructed using codons with a high frequency of use in Escherichia coli.

[0103] A nucleic acid molecule encoding an HTRA1 inhibitory peptide may be operably linked to one, two or more regulatory sequences. The expression "operably linked" is used to mean that the linked nucleic acid molecule can be expressed, or the nucleotide sequence contained in the molecule is expressed. The regulatory sequence contains a sequence element that includes information about the regulation of transcription and/or regulation of translation. Although the regulatory sequence varies by species, the regulatory sequence typically includes a promoter and includes 5' non-coding sequences involved in transcription and translation initiation, such as the prokaryotic -35/-10 box and the Shine-Dalgarno sequence or the eukaryotic TATA- box, CAAT sequence and 5'-capping sequence. Such a sequence may contain an enhancer element and/or a repressor element, and a translated signal sequence, a leader sequence, or the like to deliver the natural or mature peptide to a specific compartment inside or outside the host cell. The regulatory sequence may further include a 3' non-coding sequence, and this sequence may contain an element involved in transcription termination or polyadenylation, etc. However, a transcription termination sequence that is not functional enough in certain host cells can be replaced with a sequence that is suitable for the cells.

[0104] Examples of a promoter sequence may include the prokaryotic tet promoter, the lacUV5 promoter and the T7 promoter, and the SV40 promoter and the CMV promoter for eukaryotic cells.

[0105] The nucleic acid molecule encoding the HTRA1 inhibitory peptide may be in an isolated form or in a form contained in a vector or other cloning vehicle (hereinafter simply referred to as "vector; plasmid, phagemid, phage, baculovirus, cosmid", etc.). etc.), or in the chromosome, although the form is not limited to this. The vector may contain, in addition to the nucleotide sequence of the HTRA1 inhibitory peptide and the regulatory sequence, a replicating sequence and a control sequence suitable for the host cells to be used for expression, and a selectable marker that confers a phenotype allowing selection of cells carrying the nucleic acid molecule. through transformation or the like.

[0106] A nucleic acid molecule encoding an HTRA1 inhibitory peptide or a vector containing the nucleotide sequence of an HTRA1 inhibitory peptide can be transfected into host cells capable of expressing the peptide or nucleotide sequence by a method known to those skilled in the art, such as transformation. Host cells carrying the nucleic acid molecule or vector can be cultured under conditions suitable for expression of the peptide or nucleotide sequence. The host cells can be any prokaryotic or eukaryotic cell. Examples of prokaryotes may include E. coli and Bacillus subtilis. Examples of eukaryotic cells may include yeast cells such as Saccharomyces cerevisiae and Pichia pastoris, insect cells such as SF9 and High 5, and animal cells such as HeLa cells, CHO cells, COS and NS0 cells. The peptide of the present invention, when expressed using host cells such as eukaryotic cells, may undergo the desired post-translational modification. Examples of post-translational modification may include adding a functional group such as a sugar chain, adding a peptide or protein, and modifying the chemical properties of an amino acid. Alternatively, the peptide of the present invention may be artificially modified as desired. Such a modified form of a peptide is also included within the scope of the term "peptide" of the present invention.

[0107] The present invention also includes a method for producing an HTRA1 inhibitory peptide. The method includes: step 1 cultivating a host cell containing a nucleic acid molecule encoding an HTRA1 inhibitory peptide, or a vector containing a nucleotide sequence of an HTRA1 inhibitory peptide, or a cell expressing an HTRA1 inhibitory peptide; and/or step 2 isolating the HTRA1 inhibitory peptide from the cultures obtained in step 1. Operations such as fractionation, chromatography or purification known to those skilled in the art can be applied to step 2. For example, affinity purification using an antibody of the present invention mentioned below applies to this.

[0108] In some aspects of the present invention, the HTRA1 inhibitory peptide contains an intramolecular disulfide bond. It may be preferable that the peptide containing an intramolecular disulfide bond is delivered to a cellular compartment having a redox environment using a signal sequence or the like. The oxidizing environment may be provided by the periplasmic space of a Gram-negative bacterium such as E. coli, the extracellular environment of a Gram-positive bacterium, the lumen of the endoplasmic reticulum of a eukaryotic cell, or the like. Such an environment is capable of providing the formation of a structural disulfide bond. Alternatively, a peptide containing an intramolecular disulfide bond can be produced in the cytoplasm of a host cell, such as an E. coli cell. In this case, the peptide can be directly prepared in a soluble folded state or isolated in the form of an inclusion body and then reconstituted in vitro. In addition, a host cell having an oxidative intracellular environment is selected, and a peptide containing an intramolecular disulfide bond can also be produced in its cytoplasm. On the other hand, when the HTRA1 inhibitory peptide does not have an intramolecular disulfide bond, the peptide can be produced in a cell compartment having a reducing environment, such as the cytoplasm of a Gram-negative bacterium.

[0109] The HTRA1 inhibitory peptide of the present invention can also be obtained by other methods known to those skilled in the art, such as chemical synthesis, for example, the method of solid phase peptide synthesis, Merrifield et al. and a method for organic chemical synthesis of peptides using tert-butoxycarbonyl (Boc) or 9-fluorenylmethoxycarbonyl (Fmoc) and in vitro translation.

[0110] In some aspects, the present invention provides an antibody that binds to a mutant SPINK2 peptide having HTRA1 inhibitory activity and a functional fragment thereof. The antibody may be any of a polyclonal antibody and a monoclonal antibody. The monoclonal antibody is not particularly limited if the monoclonal antibody is or is derived from an immunoglobulin. The functional fragment of an antibody is not limited as long as the functional fragment has antigen-binding activity, i. e. binding activity against the mutant SPINK2 peptide. Examples thereof include both or one of the heavy and light chains or a fragment thereof, an antibody fragment lacking a constant region or Fc region, and a conjugate with an additional protein or labeling compound. Such an antibody and a functional fragment thereof can be prepared in a manner known to those skilled in the art and can be used, for example, to purify a mutant SPINK2 peptide by affinity chromatography, conduct a clinical study related to a pharmaceutical composition containing the peptide or use it, detect the peptide in diagnosis or the like, and in immunoassay. The antibody of the present invention can be purified by affinity chromatography using the peptide of the present invention to which the antibody binds.

[0111] 5. Pharmaceutical composition

The present invention also relates to a pharmaceutical composition containing an HTRA1 inhibitory peptide or a conjugate thereof.

[0112] A pharmaceutical composition containing an HTRA1 inhibitory peptide of the present invention or a conjugate thereof is useful for treating and/or preventing various diseases that are induced or exacerbated by HTRA1, and can suppress the induction or exacerbation, treat, control or suppress symptoms, prevent secondary disease, etc. by inhibiting or suppressing the expression or function of HTRA1 (hereinafter, these diseases are referred to as "diseases associated with HTRA1"). HTRA1 related diseases also include various diseases that can suppress induction or exacerbation, treat, control or suppress symptoms, prevent a secondary disease, and so on. through a protective effect on the retina. The HTRA1 inhibitory peptide has a retinal protective effect and is useful in the treatment and/or prevention of these HTRA1 related diseases.

[0113] Examples of diseases associated with HTRA1 may include, but are not limited to, age-related macular degeneration, retinopathy of prematurity, polypoid choroidal vasculopathy, rheumatoid arthritis, and osteoarthritis. Examples of age-related macular degeneration may include, but are not limited to, wet age-related macular degeneration, dry age-related macular degeneration, and geographic atrophy. In addition, the pharmaceutical composition of the present invention can be used as a photoreceptor cell protecting agent, a retinal protecting agent, and the like (hereinafter collectively referred to as "retinal protecting agent").

Age-related macular degeneration is divided into wet and dry forms. The wet form is further classified into typical age-related macular degeneration (typical AMD), polypoid choroidal vasculopathy (PCV), and retinal angiomatous proliferation (RAP). For the wet form, there are treatments such as anti-VEGF drugs and photodynamic therapy (PDT), which, however, are not always sufficient. For the dry form, only dietary modification or supplementation based on the Age-Related Eye Disease Study (AREDS) is practiced.

The pharmaceutical composition of the present invention can be used to treat or prevent age-related macular degeneration, for example, as follows from the results showing that: the number of nuclei in the outer nuclear layer decreased in the control group in a rat model of light-induced retinal injury, while administration of the HTRA1 inhibitory peptide of the present invention before or after exposure to light suppressed the reduction in the number of nuclei in the outer nuclear layer in the rat model administration group (examples 5, 10 and 14); and the area of retinal pigment epithelial cells (RPE cells) or the number of RPE cells having a cell area equal to or greater than a given value increased in the control group in a rabbit model of retinal injury (indicated below) obtained by feeding a high-fat diet and inclusion hydroquinone, while administration of the HTRA1 inhibitory peptide of the present invention suppressed this increase in the rabbit model administration group (Example 11). The rabbit retinal injury model takes into account the risk factors for developing dry macular degeneration in humans and is therefore an excellent model of dry age-related macular degeneration in particular.

In a VEGF mRNA induction test using retinal pigment epithelial (RPE) cells, it was confirmed that administration of an HTRA1 inhibitory peptide suppresses VEGF mRNA expression (Example 12). VEGF induction in retinal pigment epithelial cells has been implicated in the pathogenesis of wet age-related macular degeneration (Klettner A. et al. (2009) Graefes Arch. Clin. Exp. Ophthalmol., Vol. 247: p. 1487-1492). In addition, such abnormal VEGF induction is also believed to be involved in maintaining the pathological condition, indicating that the HTRA1 inhibitory peptide of the present invention is useful for the treatment and prevention of dry age-related macular degeneration and wet age-related macular degeneration.

In the human umbilical vein endothelial cell (HUVEC) migration test, administration of an HTRA1 inhibitory peptide was confirmed to inhibit migration (Example 13), indicating that the HTRA1 inhibitory peptide of the present invention is useful for the treatment and prevention of wet age-related macular degeneration, characterized by angiogenesis.

The wet form of age-related macular degeneration tends to progress. Although administration of an anti-VEGF drug may temporarily suppress the disease state, recurrence at a high rate is a problem (Yand S. et al., Drug Des. Devel. Ther., Vol. 2, No. 10: p. 1857 -67, 2016). The problem of drugs discovered in recent years is the development of atrophy and a decrease in visual acuity in patients with the wet form of age-related macular degeneration (Berg K. et al., Acta Ophthalmologica, Vol. 95, No. 8: p. 796-802, 2017 ). The administration of the HTRA1 inhibitory peptide of the present invention alone or in combination with an anti-VEGF drug may have a therapeutic effect on the wet form of age-related macular degeneration (including polypoid choroidal vasculopathy and retinal angiomatous proliferation). In addition, prophylactic administration of the peptide can prevent or delay the recurrence of the disease state and delay the initiation or resumption of treatment with, for example, an anti-VEGF drug. In addition, the peptide inhibits the development of atrophy in patients with wet age-related macular degeneration and, therefore, is able to provide long-term effects in terms of visual acuity.

The HTRA1 inhibitory peptide of the present invention is advantageous in terms of tissue penetration (Example 11), and also advantageous in terms of its physical properties, stability, safety, post-administration kinetics, productivity, etc. and thus may preferably be formulated as an active ingredient in a pharmaceutical composition.

[0114] The pharmaceutical composition of the present invention may contain a therapeutically or prophylactically effective amount of an HTRA1 inhibitory peptide or conjugate thereof, and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant.

[0115] The term "therapeutically or prophylactically effective amount" is used to refer to an amount that has a therapeutic or prophylactic effect on a particular disease when administered in a dosage form and by a certain route of administration, and is synonymous with "pharmacologically effective amount".

[0116] The pharmaceutical composition of the present invention may contain a substance to change, maintain or maintain pH, osmotic pressure, viscosity, transparency, color, isotonicity, sterility or stability, solubility, sustained release rate, absorption, penetration, dosage form, activity, properties , shapes, etc. the composition or peptide of the present invention or the conjugate contained therein (hereinafter referred to as "pharmaceutical substance"). The pharmaceutical substance is not particularly limited as long as the compound is pharmacologically acceptable. For example, no or low toxicity is a property that a pharmaceutical substance preferably has.

[0117] Examples of a pharmaceutical substance may include, but are not limited to, amino acids such as glycine, alanine, glutamine, asparagine, histidine, arginine, and lysine; antimicrobial agents; antioxidants such as ascorbic acid, sodium sulfate and sodium bisulfite; buffers such as phosphate, citrate or borate buffers, sodium bicarbonate and Tris-HCl; fillers such as mannitol and glycine; chelating agents such as ethylenediaminetetraacetic acid (EDTA); complexing agents such as caffeine, polyvinylpyrrolidine, β-cyclodextrin and hydroxypropyl-β-cyclodextrin; bulking agents such as glucose, mannose and dextrin; other carbohydrates such as monosaccharides, disaccharides, glucose, mannose and dextrin; dyes; flavors; diluents; emulsifiers; hydrophilic polymers such as polyvinylpyrrolidine; low molecular weight polypeptides; salt-forming counterions; antiseptics such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, and hydrogen peroxide; solvents such as glycerin, propylene glycol and polyethylene glycol; sugar alcohols such as mannitol and sorbitol; suspending agents; surfactants such as PEG, sorbitan ester, polysorbates such as polysorbate 20 and polysorbate 80, triton, tromethamine, lecithin and cholesterol; stability enhancing compounds such as sucrose and sorbitol; elasticity enhancers such as sodium chloride, potassium chloride, mannitol and sorbitol; transport agents; diluents: excipients; and/or pharmaceutical adjuvants.

[0118] Such a pharmaceutical substance is added to the HTRA1 inhibitory peptide in an amount exceeding the weight of the HTRA1 inhibitory peptide by 0.001-1000 times, preferably 0.01-100 times, and more preferably 0.1-10 times.

[0119] A liposome containing an HTRA1 inhibitory peptide or a conjugate thereof, or a pharmaceutical composition containing a modified form comprising an HTRA1 inhibitory peptide or a conjugate thereof conjugated to a liposome is also included in the term "pharmaceutical composition" of the present invention.

[0120] The excipient or carrier is not particularly limited as long as the excipient or carrier is generally a liquid or solid and is water for injection, saline, artificial cerebrospinal fluid, or other substances for use in oral or parenteral preparations. Examples of normal saline may include neutral normal saline or normal saline containing serum albumin.

[0121] Examples of a buffer may include Tris buffer prepared to bring the final pH of the pharmaceutical composition to 7.0-8.5, acetate buffer prepared to bring its final pH to 4.0-5.5, citrate buffer, prepared to bring its final pH to 5.0-8.0 and a histidine buffer prepared to bring its final pH to 5.0-8.0.

[0122] The pharmaceutical composition of the present invention is a solid, liquid, suspension, or the like. Another example of a pharmaceutical composition of the present invention may include lyophilized preparations. Freeze-dried preparations can be prepared using an excipient such as sucrose.

[0123] The route of administration of the pharmaceutical composition of the present invention can be any: instillation into the eyes, enteral administration, topical administration and parenteral administration. Examples thereof may include conjunctival instillation, intravitreal administration, intravenous administration, intraarterial administration, intramuscular administration, intradermal administration, subcutaneous administration, intraperitoneal administration, transdermal administration, intraosseous administration, and intraarticular administration.

[0124] The composition of the pharmaceutical composition can be determined according to the route of administration, the binding affinity of HTRA1 to the HTRA1 inhibitory peptide, etc. An HTRA1 inhibitory peptide of the present invention having a higher inhibitory activity (lower IC ₅₀ ) against the target HTRA1 or having a higher affinity (lower K _D ) for the HTRA1 protein is able to exert its therapeutic effect at a lower dose.

[0125] The dose of the HTRA1 inhibitory peptide of the present invention or its conjugate is not limited as long as the dose is a pharmacologically effective amount. The dose may be appropriately determined based on the subject's species, disease type, symptoms, sex, age, pre-existing pathological conditions, the binding affinity of the peptide to the HTRA1 protein or its biological activity, and other factors. The dose is usually 0.01-1000 mg/kg and preferably 0.1-100 mg/kg, it can be administered from once a day to once every 180 days, or two or three or more times a day.

[0126] Examples of the form of the pharmaceutical composition may include injectable preparations (including lyophilized preparations and drip infusions), suppositories, absorption-type preparations for transnasal administration, absorption-type preparations for transdermal administration, sublingual agents, capsules, tablets, ointments, granules, aerosols, pills , powders, suspensions, emulsions, eye drops and biological implant formulations.

[0127] A pharmaceutical composition containing an HTRA1 inhibitory peptide or a conjugate thereof as an active ingredient can be administered simultaneously or separately from an additional drug. For example, a pharmaceutical composition containing an HTRA1 inhibitory peptide or a conjugate thereof as an active ingredient may be administered after administration of an additional drug, or an additional drug may be administered after administration of the pharmaceutical composition. Alternatively, the pharmaceutical composition and the additional drug may be administered simultaneously. For simultaneous administration, the HTRA1 inhibitory peptide or conjugate thereof and the additional drug may be in the same formulation or may be in separate formulations (multiple formulations).

[0128] Examples of an additional drug used in combination with the pharmaceutical composition of the present invention may include anti-VEGF agents, anti-inflammatory agents, agents that neutralize inflammatory cytokines, and inhibitors of the complement activation pathway. Anti-VEGF agents are classified into anti-VEGF antibodies, VEGF inhibitors, VEGF receptor antagonists and soluble VEGF receptors, etc. and include bevacizumab, ranibizumab, aflibercept, pegaptanib, brolucizumab and the like. The anti-inflammatory agent is not particularly limited as long as the anti-inflammatory agent can be locally administered to suppress intraocular or intraarticular inflammation. Examples of an inflammatory cytokine neutralizing agent include anti-TNFα antibodies, anti-interleukin-6 (hereinafter referred to as "IL-6") antibodies, anti-IL-6 receptor antibodies, and soluble TNF receptors, and may specifically include infliximab, adalimumab , golimumab, certolizumab, tocilizumab and etanercept. Examples of a complement pathway inhibitor may include lampalizumab. These drugs are suitable for the treatment or prevention of diseases associated with HTRA1, and can also be combined with the pharmaceutical composition of the present invention in the treatment or prevention of diseases other than those associated with HTRA1.

[0129] You can use one of these additional drugs, or two, three or more of them can be entered or received. Such approaches are collectively referred to as "combination with an additional drug" or "combination with an additional drug" and the pharmaceutical composition of the present invention. A pharmaceutical composition of the present invention containing an additional drug in addition to the antibody of the present invention, its binding fragment or a modified form of the antibody or fragment, or used in combination with additional therapy, is also included in the present invention as an aspect of the invention "combination use with additional drug” or “combination with an additional drug”.

[0130] The present invention also relates to a method for treating or preventing a disease associated with HTRA1, such as age-related macular degeneration, comprising the step of administering an HTRA1 inhibitory peptide or a conjugate thereof, using the HTRA1 inhibitory peptide of the present invention or a conjugate thereof to prepare a pharmaceutical composition for the treatment or prevention of a disease, and the use of an HTRA1 inhibitory peptide or conjugate thereof for the treatment or prevention of a disease. The present invention also includes a treatment or prophylaxis kit containing an HTRA1 inhibitory peptide of the present invention or a conjugate thereof.

[0131] The present invention further relates to a polynucleotide comprising a nucleotide sequence encoding the amino acid sequence of an HTRA1 inhibitory peptide or a conjugate thereof, a vector containing the polynucleotide, and a pharmaceutical composition comprising the polynucleotide or vector, or a cell expressing the HTRA1 inhibitory peptide of the present invention or its conjugate. For example, the polynucleotide and the vector can be used for gene therapy of diseases associated with HTRA1 using a known approach. The cell can be used for cell therapy of diseases associated with HTRA1 using a known approach. Also, the polynucleotide or vector can be transfected, for example, autologous cells or allogeneic cells (homologous cells) to prepare cells for cell therapy. Such a polynucleotide and a vector are also included in the cell therapy drug compositions of the present invention. However, the form of the pharmaceutical composition of the present invention, including a polynucleotide, a vector, a cell, or the like, is not limited to the above.

[0132] 6. Diagnostic composition and methods for detecting and isolating HTRA1

The HTRA1 inhibitory peptide of the present invention or a conjugate thereof can have an HTRA1 binding activity in addition to an HTRA1 protease inhibitory activity, and can be used in various assays such as use as a positive control in HTRA1 inhibitor studies, HTRA1 detection, screening, and diagnostics using detection, separation of HTRA1, as reagents and other purposes. For detection or separation of HTRA1, at least one of the peptide of the present invention and HTRA1 can be immobilized.

The present invention provides a detection or diagnostic composition (hereinafter referred to collectively as a "diagnostic composition") comprising a peptide of the present invention that binds to HTRA1, or a conjugate thereof.

The diagnostic composition of the present invention is useful in screening or diagnosing diseases associated with HTRA1, HTRA1 expression, etc. In the present invention, examples of examination or diagnosis include, but are not limited to, determining or assessing the risk of developing a disease, determining the presence or absence of a disease, assessing the degree of progression or exacerbation, assessing or determining the effectiveness of treatment with a pharmaceutical composition containing an HTRA1 inhibitory peptide or its conjugate, assessing or determining the effectiveness of treatment other than this therapy, assessing the risk of recurrence and determining the presence or absence of relapse.

The diagnostic composition of the present invention is useful in identifying a recipient subject for a peptide of the present invention or a conjugate thereof, a composition comprising a peptide or a conjugate thereof, or a pharmaceutical composition comprising a peptide or a conjugate thereof.

The diagnostic composition may contain a pH buffer, an osmotic pressure regulator, salts, a stabilizer, an antiseptic, a developer, a sensitizer, an anti-aggregation agent, and the like.

The present invention also relates to a method for screening or diagnosing a disease associated with HTRA1, using a peptide of the present invention to prepare a composition for diagnosing a disease, and using a peptide of the present invention that binds to HTRA1 or a conjugate thereof for screening or diagnosing a disease. The present invention also includes an examination or diagnostic kit comprising the peptide of the present invention or a conjugate thereof.

The screening or diagnostic method comprising the peptide of the present invention that binds to HTRA1 is desirably a sandwich-type ELISA. Alternatively, a conventional detection method such as ELISA, RIA, ELISPOT, dot blot, Ouchterlony, CIE, CLIA, or flow cytometry can be used. Examination or diagnosis is also achieved in a manner based on the immunoprecipitation method.

The present invention also provides a method for detecting or measuring HTRA1 in a test sample. In such a detection or measurement method, the diagnostic composition of the present invention can be used. HTRA1 in a test sample can be detected by: contacting an HTRA1 inhibitory peptide or conjugate thereof with a test sample (step 1); and subsequent measurement of the amount of HTRA1 associated with the peptide (step 2). Step 1 may include, for example, immobilizing the HTRA1 inhibitory peptide conjugate with the immunoglobulin Fc region on magnetic beads through the G protein and adding the test sample thereto. Step 2 may include, for example, separating the magnetic beads and analyzing the soluble protein precipitated with the beads using SDS-PAGE or Western blotting to detect HTRA1. In addition to a human or non-human animal sample, even an artificially processed sample, such as a recombinant protein, can be subjected to this measurement. Examples of a test sample from a subject's body may include, but are not limited to, blood, synovial fluid, ascitic fluid, lymph, cerebrospinal fluid, bronchoalveolar lavage, saliva, sputum, tissue homogenate supernatants, and tissue sections.

Detection of HTRA1 can be carried out not only in vitro, but also in vivo. In the case of diagnostic imaging, an HTRA1 inhibitory peptide or conjugate thereof labeled with a pharmaceutically acceptable radionuclide or light emitter can be used. Step 1 may include, for example, administering the labeled peptide or conjugate thereof to the subject to be tested. Step 2 may include, for example, obtaining an image using a diagnostic imaging technique such as PET/CT and determining or examining the presence of HTRA1.

The peptide or conjugate thereof contained in the diagnostic composition of the present invention binds to HTRA1 and preferably has HTRA1-specific binding activity.

The present invention also includes a method for identifying a recipient subject for a pharmaceutical composition of the present invention. In such a method for identifying HTRA1 in a sample obtained from a subject, it is measured using the HTRA1 binding peptide of the present invention, and the subject can be determined as positive when HTRA1 is detected in the sample or when more HTRA1 is detected in it compared to the amount of HTRA1, detected in a sample obtained from a healthy subject. In this method, the diagnostic composition of the present invention can be used.

In a preferred aspect of the method of identification, the subject suffers from a disease associated with HTRA1, or is at risk of developing such a disease.

In one aspect, the pharmaceutical composition of the present invention may be administered to a subject determined to be positive in the identification method.

[0133] HTRA1 can be specifically separated from a sample in which HTRA1 is found together with other components using a peptide of the present invention having an HTRA1-specific binding activity, or a conjugate thereof. Release of HTRA1 from a peptide can be performed non-selectively, eg, in the presence of relatively high ionic strength, low pH, moderate denaturation conditions, or a chaotropic salt, and is preferably performed without impairing the protease activity of HTRA1.

[0134] 7. A method for identifying a therapeutic agent or prophylactic agent for a disease associated with HTRA1.

In one aspect, the present invention relates to a method for identifying a therapeutic agent or prophylactic agent for an HTRA1 related disease, and preferably age-related macular degeneration, or a candidate thereof, using HTRA1 inhibitory activity as an index. The method may include: step 1 incubation of the HTRA1 protease and the substrate in the presence or absence of the test substance (or in the presence of a carrier); step 2 determining HTRA1 protease activity in the presence and absence of the test substance; and/or step 3 of determining the test substance as a therapeutic or prophylactic agent for age-related macular degeneration or a candidate thereof, when the activity of the HTRA1 protease in the presence of the test substance is lower than the activity of the HTRA1 protease in the absence of the test substance. The test substance may be peptide or non-peptide. The peptide test substance is not limited to SPINK2 mutants. Examples thereof may include, but are not limited to, antibodies, peptides other than SPINK2 mutants having a protein backbone other than immunoglobulin, and analogs of HTRA1 substrates. Examples of a non-peptide test substance may include, but are not limited to, synthetic small molecule compounds and nucleic acids. One or two or more of the steps described above may also preferably be included in a method for identifying a retinal protective agent or candidate thereof. The present invention also relates to a method for identifying a retinal protective agent or a candidate thereof.

[0135] 8. Rabbit retinal injury model

The present invention also provides a rabbit model of retinal injury caused by feeding a high fat diet (hereinafter referred to as "HFD") including hydroquinone (hereinafter referred to as "HQ"), a method for obtaining a model, a method for testing using a model, etc.

[0136] For this model, an arbitrary white rabbit, such as NZW or JW, 2 years of age or older, regardless of gender, can be used.

[0137] The HFD may contain an arbitrary amount of an arbitrary fat or oil, for example, from 0.1 to 2% (w/v) cholesterol, from 1 to 10% (w/v) coconut oil, from 1 to 10 % (w/v) peanut oil, 1 to 10% (w/v) soybean oil, 10 to 20% (w/v) beef tallow, 10 to 50% (w/v) vol.) pork fat or from 1 to 10% (wt./vol.) corn oil, although the components of the diet are not limited to this, provided that the components are suitable for reproducing the model.

[0138] The amount of HQ contained in HQ-HFD is 0 to 4% (w/v), preferably 0.5 to 3.5% (w/v), more preferably 1.0 up to 3.0% (w/v), even more preferably 2.2 to 2.8% (w/v) and most preferably 2.4% (w/v).

[0139] The HQ-HFD nursing period is 3 to 6 months, preferably 3.5 to 5 months, and more preferably 4 months. Continued feeding of the diet for 8 months or more should be avoided.

[0140] The model of the present invention is preferred because the model is more suitable for humans in terms of eyeball size and function compared to mouse models (Non-Patent Documents 18 and 19). For conventional rabbit models (non-patent document 20), it takes 8 months to reproduce them. On the contrary, the model of the present invention can be obtained in a shorter period, and moreover, it is more preferable as an age-related macular degeneration model because RPE cell hypertrophy can be induced in the model, which is uncertain in conventional models.

The retinal pigment epithelial cells (RPE cells) in the model of the present invention are hypertrophied compared to the RPE cells in a normal rabbit, which makes it possible to detect the early development of the pathological condition of age-related macular degeneration. On the other hand, hypertrophy of RPE cells could not be visually confirmed when 10-week-old rabbits representing a conventional rabbit model (Non-Patent Document 20) were fed HQ-HFD for 4 months.

A therapeutic agent and/or prophylactic agent for age-related macular degeneration or a retinal protective agent can be identified using this model, allowing the detection of a pathological condition. The method of identification may include the following steps: (i) assessing hypertrophy of retinal pigment epithelial cells in a rabbit in this model with or without administration of a test substance; and (ii) defining a test substance as positive when retinal pigment epithelial cell hypertrophy is suppressed when the test substance is administered compared to retinal pigment epithelial cell hypertrophy without the test substance. The evaluation in step (i) is preferably a measurement of the mean area of the retinal pigment epithelium cells and/or the number of hypertrophied retinal pigment epithelial cells.

Examples

[0141] Hereinafter, some aspects of the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.

[0142] In the following examples, unless otherwise indicated, individual genetic manipulation operations were performed according to the methods described in Molecular Cloning (Sambrook, J., Fritsch, EF and Maniatis, T., published Cold Spring Harbor Laboratory Press. in 1982 and 1989) or other methods described in the experimental guidelines used by those skilled in the art, or where commercially available reagents or kits were used, the examples were performed according to the instructions included in the commercially available products.

[0143] Example 1 Preparation of an HTRA1 inhibitory peptide

(1-1) Construction of the HTRA1 inhibitory peptide expression vector

(1-1-1) Construction of pET 32a (modified)_HTRA1 inhibitory peptide

First, an HTRA1 inhibitory peptide expression vector was constructed, with a SPINK2 scaffold as the backbone. The inhibitor fragment was amplified by PCR ((94°C for 15 s, 60°C for 30 s and 68°C for 30 s) × 30 cycles) using the nucleotide sequence (SEQ ID NO: 4, 6, 8 , 10, 12, 14, 16, 18, 20 and 22) of each inhibitory peptide and nucleotide sequence (SEQ ID NO: 2) of SPINK2 as templates using the following primers and KOD-plus - (Toyobo Co., Ltd.).

Primer 1: 5'-AAAAGAATTCTGATCCGCAGTTTGGTCTGTTTAG-3'

Primer 2: 5'-AAAACTCGAGTTATGCGGCCGCAGACGCGCCGCACGGACC-3'

Each amplified fragment was subjected to agarose gel electrophoresis. Then, the desired DNA fragment was excised from the gel, and DNA was obtained using a QIAquick DNA gel isolation kit (Qiagen N.V.). The resulting DNA fragment and pET 32a (modified) were each treated with EcoRI (New England BioLabs Inc.) and XhoI (New England BioLabs Inc.) at 37° C. for 1 hour or more. After agarose gel electrophoresis, the desired DNA fragments were excised from the gel and purified using the QIAquick PCR Purification Kit (Qiagen N.V.). The purified fragments were reacted overnight at 16°C for ligation using T4 DNA ligase (New England BioLabs Inc.). The ligation solution was added to E. coli JM109 (Toyobo Co., Ltd.) and the mixture was kept on ice for 30 minutes, then heat treated at 42°C for 45 seconds, then kept on ice for 5 minutes, and inoculated into a plate with 2YT medium containing 0.1 mg/ml ampicillin, followed by overnight static culture at 37° C. to transform E. coli. The next day, the transformed E. coli was inoculated into Terrific broth medium (Invitrogen Corp.) containing 0.1 mg/ml ampicillin and cultured overnight at 37°C. Then, plasmid DNA was isolated using the QIAprep 96 Turbo Miniprep kit (Qiagen N.V.) (hereinafter referred to as "miniprep treatment") and subjected to sequence analysis to construct "pET 32a (modified)_HTRA1 inhibitory peptide".

(1-1-2) Construction of pET 32a_HTRA1 inhibitory peptide_Kex2

Similarly, the inhibitor fragment was amplified by PCR ((94°C for 15 s, 60°C for 30 s and 68°C for 30 s) × 30 cycles) using the sequence (sequence listing) of each inhibitor and the nucleotide sequence SPINK2 as templates using the following primers and KOD-plus - (Toyobo Co., Ltd.).

Primer 3: 5'-AAAAGGATCCCTGGACAAACGTGATCCGCAGTTTGGTCTGTTTAG-3'

Primer 4: 5'-AAAACTCGAGTTAGCCGCCGCACGGACCATTGCGAATAATTTTA-3'

Each amplified fragment was subjected to agarose gel electrophoresis. Then, the desired DNA fragment was excised from the gel, and DNA was obtained using a QIAquick DNA gel isolation kit (Qiagen N.V.). The resulting DNA fragment and pET 32a (Novagen) were each treated with BamHI (New England BioLabs Inc.) and XhoI (New England BioLabs Inc.) at 37° C. for 1 hour or more. After agarose gel electrophoresis, the desired DNA fragments were excised from the gel and purified using the QIAquick PCR Purification kit (Qiagen N.V.). The purified fragments were reacted overnight at 16°C for ligation using T4 DNA ligase (New England BioLabs Inc.). The ligation solution was added to E. coli JM109 (Toyobo Co., Ltd.) and the mixture was kept on ice for 30 minutes, then heat treated at 42°C for 45 seconds, then kept on ice for 5 minutes, inoculated into a plate with 2YT medium containing 0.1 mg/ml ampicillin, followed by overnight static culture at 37° C. to transform E. coli. Transformed E. coli was cultured and then miniprep and sequence analysis were performed to construct "pET 32a_HTRA1 inhibitory peptide_Kex2". The operation was performed according to the method described in (1-1-1).

(1-2) Expression and purification of HTRA1 inhibitory peptide

E. coli Origami B (DE3) (Novagen) was transformed with the pET 32a (modified)_HTRA1 inhibitory peptide vector constructed in Example (1-1-1) and cultured at 37°C using 2YT medium containing 0.1 mg /ml ampicillin. Then IPTG (final concentration: 1 mM) was added thereto, and E. coli was cultured overnight at 16°C. The day after collection by centrifugation (3000 g, 20 min, 4°C), a lysate was prepared using BugBuster Master Mix (Novagen) and the His-tagged fusion protein of interest was purified using TALON metal chelate affinity resin (Clontech Laboratories, Inc.). The thioredoxin tag and desired protein were then digested using the Thrombin Cleavage Capture Kit (Novagen) and purified using TALON. The resulting product was subjected to gel filtration (Superdex 75 10/300 GL) or reverse phase chromatography (YMC-Pack ODS-AM) to obtain an HTRA1 inhibitory peptide. The resulting peptide was conjugated at its N-terminus to a fragment consisting of an S-tag and a linker (SEQ ID NO: 31: FIG. 43) and at its C-terminus to a C-terminal hexamer (SEQ ID NO: 32: FIG. 44) instead of Gly-Gly.

[0144] Similarly, E. coli Origami B (DE3) (Novagen) was transformed with the pET 32a_HTRA1 inhibitory peptide Kex2 vector constructed in Example (1-1-2) and cultured at 37°C using 2YT medium containing 0, 1 mg/ml ampicillin. Then IPTG (final concentration: 1 mM) was added thereto, and E. coli was cultured overnight at 16°C. The day after collection by centrifugation (3000 g, 20 min, 4°C), a lysate was prepared using BugBuster Master Mix (Novagen) and the His-tagged fusion protein of interest was purified using TALON TALON metal chelate affinity resin ( Clontech Laboratories, Inc.). Then, the thioredoxin tag and the desired protein were digested using Kex2 (Saccharomyces cerevisiae: accession number CAA96143) and purified using TALON. The resulting product was subjected to gel filtration (Superdex 75 10/300 GL) or reverse phase chromatography (YMC-Pack ODS-AM) to obtain an HTRA1 inhibitory peptide (neither N-terminus nor C-terminus conjugated with a label, linker or the like).

[0145] Example 2 Evaluation of an HTRA1 inhibitory peptide for HTRA1 inhibitory activity

The sequence similarity of human, mouse, rat and monkey HTRA1 is shown in FIG. 1. The primary sequence constituting the HTRA1 protease domain (204Gly to 364Leu), which is the enzymatically active domain, is completely identical between human and monkey. The sequences of the protease domains of human, mouse or rat HTRA1 differ by 1 residue. However, this residue is structurally located at the site opposite the active site of the enzyme and, therefore, presumably does not affect the active site of the enzyme (Fig. 1). Therefore, the protease domain of HTRA1 has almost the same sequence regardless of the species (human/mouse/rat/monkey). Thus, no specific conclusions regarding species have been made.

(2-1) Preparation of the HTRA1 protease domain of HTRA1 (catalyte)

(2-1-1) Construction of pET 21b_HTRA1 (catalyte)

The protease domain (158Gly to 373Lys), excluding the N-terminal domain and PDZ domain, of human HTRA1 (Q92743) was used as HTRA1 (catalyte) to construct the HTRA1 expression vector (catalyte). The desired DNA fragment was amplified by PCR ((94°C for 15 s, 60°C for 30 s and 68°C for 45 s) × 30 cycles) using a human HTRA1 inserted plasmid (GeneCopoeia, Inc.; GC-M0558) as a template using the following primers and KOD-plus - (Toyobo Co., Ltd.).

Primer 5: 5'-AAACATATGGGGCAGGAAGATCCCAACAGTTTGC-3'

Primer 6: 5'-AAACTCGAGTTTGGCCTGTCGGTCATGGGACTC-3'

The amplified fragment was subjected to agarose gel electrophoresis. Then, the desired DNA fragment was excised from the gel, and DNA was obtained using a QIAquick DNA gel isolation kit (Qiagen N.V.). The resulting DNA fragment and pET 32a (Novagen) were each treated with NdeI (New England BioLabs Inc.) and XhoI (New England BioLabs Inc.) at 37° C. for 1 hour or more. After agarose gel electrophoresis, the desired DNA fragments were excised from the gel and purified using the QIAquick PCR Purification Kit (Qiagen N.V.). The purified fragments were reacted overnight at 16°C for ligation using T4 DNA ligase (New England BioLabs Inc.). The ligation solution was added to E. coli JM109 (Toyobo Co., Ltd.) and the mixture was kept on ice for 30 minutes, then heat treated at 42°C for 45 seconds, then kept on ice for 5 minutes and inoculated in a plate with 2YT medium containing 0.1 mg/ml ampicillin, followed by overnight static culture at 37°C to transform E. coli. Transformed E. coli was cultured and then miniprep and sequence analysis were performed to construct "pET 21b_HTRA1 (catalyte)". The operation was performed according to the method described in Example (1-1-1).

(2-1-2) Preparation of HTRA1 (catalyte)

E. coli BL21 (DE3) (Novagen) was transformed with engineered pET 21b_HTRA1 (catalyte) and cultured at 37° C. using 2YT medium containing 0.1 mg/ml ampicillin. IPTG was then added thereto (final concentration: 1 mM) and E. coli was cultured overnight at 28°C. After collection, the lysate was prepared by suspending in phosphate buffer (50 mM sodium phosphate and 300 mM NaCl) containing 1 mg/ml lysozyme and sonicating, and the desired His-tagged fusion protein was isolated using TALON (Clontech Laboratories, Inc.). The resulting product was subjected to gel filtration (Superdex 200 10/300 GL) to purify HTRA1 (catalyte).

(2-2) Acquisition of full size HTRA1 (HTRA1 (full))

(2-2-1) Construction of pcDNA3.1_HTRA1 (complete)_His

The desired DNA fragment was amplified by PCR ((94°C for 15 s, 60°C for 30 s and 68°C for 90 s) × 30 cycles) using synthesized human HTRA1 DNA (Q92743) (GeneArt) in as a template using the following primers and KOD-plus - (Toyobo Co., Ltd.).

Primer 7: 5'-AAAAGAATTCGCCACCATGCAGATTCCTAGAGCCG-3'

Primer 8: 5'-AAAACTCGAGTCAGTGGTGATGGTGGTGGTGGCCGG-3'

The amplified fragment was subjected to agarose gel electrophoresis. Then, the desired DNA fragment was excised from the gel, and DNA was obtained using a QIAquick DNA gel isolation kit (Qiagen N.V.). The resulting DNA fragment and pcDNA3.1 (Thermo Fisher Scientific Inc.) were each treated with EcoRI (New England BioLabs Inc.) and XhoI (New England BioLabs Inc.) at 37° C. for 1 hour or more. After agarose gel electrophoresis, the desired DNA fragments were excised from the gel and purified using the QIAquick PCR Purification Kit (Qiagen N.V.). The purified fragments were reacted overnight at 16°C for ligation using T4 DNA ligase (New England BioLabs Inc.). The ligation solution was added to E. coli JM109 (Toyobo Co., Ltd.) and the mixture was kept on ice for 30 minutes, then heat treated at 42°C for 45 seconds, then kept on ice for 5 minutes and inoculated into a plate with 2YT medium containing 0.1 mg/ml ampicillin, followed by overnight static culture at 37° C. to transform E. coli. Transformed E. coli was cultured and then miniprep and sequence analysis were performed to construct "pcDNA3.1_HTRA1 (full)_His". The operation was performed according to the method described in Example (1-1-1).

(2-2-2) Construction of pcDNA3.3_HTRA1 (full)_FLAG_His

Fragment A was amplified by PCR ((94°C for 15 s, 60°C for 30 s and 68°C for 90 s) × 30 cycles) using pcDNA3.1_HTRA1 (complete)_His constructed in example ( 2-2-1) as a template using the following primers and KOD-plus - (Toyobo Co., Ltd.).

Primer 7

Primer 9: 5'-CTTGTCGTCATCGTCCTTGTAGTCGCCGGGGTCGATTCCTC-3'

Fragment B was then amplified by PCR ((94°C for 15 s, 60°C for 30 s and 68°C for 90 s) × 30 cycles) using the following primers and KOD-plus - (Toyobo Co. , Ltd.).

Primer 10: 5'-GCGACTACAAGGACGATGACGACAAGCACCACCACCATCATCAC-3'

Primer 11: 5'-AAAAACTCGAGCTAGTGATGATGGTGGTGGTGCTTGTCGTC-3'

The desired DNA fragment was amplified by PCR ((94°C for 15 s, 60°C for 30 s and 68°C for 90 s) × 30 cycles) using fragment A and fragment B as templates using primers 7 and 11 and KOD-plus - (Toyobo Co., Ltd.). The amplified fragment was subjected to agarose gel electrophoresis. Then, the desired DNA fragment was excised from the gel, and DNA was obtained using a QIAquick DNA gel isolation kit (Qiagen N.V.). The resulting DNA fragment and pcDNA3.3 (Thermo Fisher Scientific Inc.) were each treated with EcoRI (New England BioLabs Inc.) and XhoI (New England BioLabs Inc.) at 37° C. for 1 hour or more. After agarose gel electrophoresis, the desired DNA fragments were excised from the gel and purified using the QIAquick PCR Purification Kit (Qiagen N.V.). The purified fragments were reacted overnight at 16°C for ligation using T4 DNA ligase (New England BioLabs Inc.). The ligation solution was added to E. coli JM109 (Toyobo Co., Ltd.) and the mixture was kept on ice for 30 minutes, then heat treated at 42°C for 45 seconds, then kept on ice for 5 minutes and inoculated into a plate with 2YT medium containing 0.1 mg/ml ampicillin, followed by overnight static culture at 37° C. to transform E. coli. Transformed E. coli was cultured and then miniprep and sequence analysis were performed to construct "pcDNA3.3_HTRA1 (full)_FLAG_His". The operation was performed according to the method described in Example (1-1-1).

(2-2-3) Get HTRA1 (complete)

FreeStyle 293F cells (Thermo Fisher Scientific Inc.) were transfected with pcDNA3.3_HTRA1 (total)_FLAG_His constructed in Example (2-2-2) using polyethyleneimine Max (Polysciences, Inc.). Six days later, the culture supernatant was isolated. The His-tagged fusion protein was isolated using a HisTrap excel column (GE Healthcare) and HTRA1 (total) was further purified using an anti-FLAG M2 affinity gel (Sigma-Aldrich Co. LLC).

[0146] (2-3) Preparation of inactive HTRA1, mutant HTRA1 (S328A)

(2-3-1) Construction of pcDNA3.3_HTRA1 (S328A)_FLAG_His

To construct the expression vector of inactive HTRA1, mutant HTRA1 (S328A), PCR ((95°C for 30 s, 55°C for 1 min and 68°C for 7 min)) × 18 cycles was performed using the vector “pcDNA3 .3_HTRA1 (full)_FLAG_His" constructed in Example (2-2-2) as a template using the following primers and QuikChange II site-directed mutagenesis kits (Agilent Technologies Japan, Ltd.).

Primer 21: 5'-CCATCATCAACTACGGCAACGCGGGCGGACCCCTCGTGAACC-3' (SEQ ID NO: 55: Fig. 76)

Primer 22: 5'-GGTTCACGAGGGGTCCGCCCGCGTTGCCGTAGTTGATGATGG-3' (SEQ ID NO: 56: Fig. 77)

After the PCR reaction, E. coli JM109 (Toyobo Co., Ltd.) was transformed with the PCR reaction solution treated with DpnI following the protocol supplied with the kit. Transformed E. coli was cultured and then miniprep and sequence analysis were performed to construct "pcDNA3.3_HTRA1(S328A)_FLAG_His". The operation was performed according to the method described in Example (1-1-1).

(2-3-2) Obtaining HTRA1 (S328A)

HTRA1 (S328A) was expressed using FreeStyle 293F cells according to the method described in Example (2-2-3), and HTRA1 (S328A) was obtained by affinity purification.

Example 3 Evaluation of an HTRA1 inhibitory peptide for HTRA1 inhibitory activity

(3-1) Evaluation of an HTRA1 inhibitory peptide for HTRA1 inhibitory activity using a peptide substrate

Peptide substrate H2-Opt (Mca-IRRVSYSFK (Dnp) K) (Peptide Institute, Inc.: SEQ ID NO: 54, Fig. 8) was dissolved at 10 mM in DMSO, diluted with assay buffer (50 mM borate and 150 mM NaCl , pH 8.5) and used at a final concentration of 10 μM. HTRA1 (HTRA1 (catalyte) or HTRA1 (total)) and each HTRA1 inhibitory peptide diluted with assay buffer were mixed at 25 μl each and reacted at 37°C for 20 min. Then, 50 μl of substrate diluted with assay buffer was added to the mixture. Fluorescent signal (excitation wave 328 nm/emission wave 393 nm) was measured using an Enspire (PerkinElmer, Inc.). The final concentration of HTRA1 was 100 nM and the final concentration of HTRA1 inhibitory peptide was 1.875 to 1000 nM. A black PROTEOSAVE® R9696 plate (Sumitomo Bakelite Co., Ltd.) was used in the reaction and measurement.

[0147] The rate of degradation of the peptide substrate in the presence of the HTRA1 inhibitory peptide at each concentration was calculated. When the cleavage rate at 0 nM inhibitor concentration was taken as 100%, the inhibition activity of HTRA1 (catalyte) and HTRA1 (total) was evaluated for each HTRA1 inhibitory peptide (FIGS. 2 and 3). As a result of calculating the 50% inhibitory concentration (IC ₅₀ ) using GraphPad Prism (version 5.0; GraphPad Software Inc.), it was found that all HTRA1 inhibitory peptides inhibit the activity of the HTRA1 enzyme (catalyte) and HTRA1 (total) at a low concentration (Fig. 2A-2C and Fig. 3). As a control: wild-type SPINK2 did not exhibit HTRA1 inhibitory activity (FIG. 2D).

[0148] HTRA1 inhibition activity of HTRA1 inhibitory peptide

[0149]

Table 1 ID IC50 (nM) for HTRA1 (catalyte) IC50 (nM) for HTRA1 (full) H218 72 55 H223 41 66 H228 71 38 H308 37 15 H321 48 29 H322 50 17 H308AT 49 31 H321AT 46 eighteen H322AT 45 25 M7 43 24

[0150]

(3-2) Evaluation of HTRA1 inhibitory peptide for HTRA1 inhibitory activity using a protein substrate

The HTRA1 inhibitory activity of the HTRA1 inhibitory peptide was evaluated with human vitronectin as a protein substrate. HTRA1 (catalyte) and each HTRA1 inhibitory peptide diluted with assay buffer (50 mM Tris and 150 mM NaCl, pH 8.0) were mixed and reacted at 37°C for 1 h. Human vitronectin ( BD Biosciences; 354238) diluted with assay buffer and reacted at 37°C for 2 hours. SDS sample buffer was added to the mixture and the enzyme reaction was stopped by treatment at 99°C for 5 minutes. The digestion of human vitronectin was then assessed by SDS-PAGE and Western blotting. The final concentration of HTRA1 inhibitory peptide was 0 to 25 μM, the final concentration of HTRA1 (catalyte) was 1 μM, and the final concentration of human vitronectin was 1 μM. For Western blotting, an anti-human vitronectin antibody (R&D Systems, Inc.; MAB2349) was used as the primary antibody, and an anti-mouse sheep IgG complete Ab conjugated to HRP (GE Healthcare; NA931) was used as the secondary antibody.

[0151] As in Example (3-1), the HTRA1 inhibitory peptides also clearly showed inhibition of HTRA1 (catalyte) when human vitronectin was used as a substrate (FIG. 4).

(3-3) Evaluation of HTRA1 inhibitory peptide for specificity

Specificity for other proteases was evaluated using cleavage of the peptide substrate as an indicator. Similarly as described in Example (3-1), each protease and each sample (final concentration: 1 μM) diluted with assay buffer were mixed at 25 μl each and reacted at 37°C for 20 minutes. Then, 50 µl of each substrate diluted with assay buffer was added to the mixture. Fluorescent signal (excitation wave 380 nm/emission wave 460 nm) was measured using Enspire (PerkinElmer, Inc.). The same assay buffer (50 mM borate and 150 mM NaCl, pH 8.5) as in Example 2 was used to assess HTRA2 activity. Assay buffer (50 mM Tris and 150 mM NaCl, pH 8.0) was used for assessment of the activity of proteases other than the activity of HTRA2. A black PROTEOSAVE® R9696 plate (Sumitomo Bakelite Co., Ltd.) was used in the reaction and measurement. The combinations of protease and substrate used in the evaluation of specificity were as follows.

Evaluation of inhibitory activity for bovine trypsin: 5 nM (final concentration) trypsin (Pierce; 20233) and 100 μM (final concentration) Boc-VPR-AMC peptide substrate, fluorogenic peptide substrate (R&D Systems, Inc.; ES011).

Evaluation of inhibitory activity for bovine α-chymotrypsin: 10 nM (final concentration) chymotrypsin (Worthington Biochemical Corporation; LS001434) and 100 μM (final concentration) Suc-Leu-Leu-Val-Tyr-MCA peptide substrate (Peptide Institute, Inc.; 3120-v).

Evaluation of inhibitory activity for human tryptase: 1 nM (final concentration) tryptase (Sigma-Aldrich Co. LLC; T7063) and 100 μM (final concentration) Boc-Phe-Ser-Arg-MCA peptide substrate (Peptide Institute, Inc.; 3107 -v).

Evaluation of inhibitory activity for human chymase: 100 nM (final concentration) chymase (Sigma-Aldrich Co. LLC; C8118) and 100 μM (final concentration) Suc-Leu-Leu-Val-Tyr-MCA peptide substrate (Peptide Institute, Inc. ; 3120-v).

Evaluation of inhibitory activity for human plasmin: 50 nM (final concentration) plasmin (Sigma-Aldrich Co. LLC; P1867) and 100 μM (final concentration) Boc-Val-Leu-Lys-MCA peptide substrate (Peptide Institute, Inc.; 3104 -v).

Evaluation of inhibitory activity for human thrombin: 1 nM (final concentration) thrombin (Sigma-Aldrich Co. LLC; T6884) and 100 μM (final concentration) Boc-VPR-AMC peptide substrate, fluorogenic peptide substrate (R&D Systems, Inc. ;ES011).

Evaluation of inhibitory activity for human matriptase: 1 nM (final concentration) matriptase (R & D Systems, Inc.; E3946-SE) and 100 μM (final concentration) Boc-QAR-AMC peptide substrate, fluorogenic peptide substrate (R & D Systems , Inc.; ES014).

Evaluation of inhibitory activity for human protein C: 100 nM (final concentration) protein C (Sigma-Aldrich Co. LLC; P2200) and 100 μM (final concentration) Boc-Leu-Ser-Thr-Arg-MCA peptide substrate (Peptide Institute, Inc.; 3112-v).

Evaluation of inhibitory activity for human tPA: 10 nM (final concentration) tPA (Sigma-Aldrich Co. LLC; T0831) and 100 μM (final concentration) Pyr-Gly-Arg-MCA peptide substrate (Peptide Institute, Inc.; 3145-v ).

Evaluation of inhibitory activity for human uPA: 10 nM (final concentration) uPA (Sigma-Aldrich Co. LLC; T0831) and 100 μM (final concentration) Pyr-Gly-Arg-MCA peptide substrate (Peptide Institute, Inc.; 3145-v )

Evaluation of inhibitory activity for human plasma kallikrein: 0.125 µg/mL (final concentration) plasma kallikrein (Sigma-Aldrich Co. LLC; T0831) and 100 µM (final concentration) Z-Phe-Arg-MCA peptide substrate (Peptide Institute, Inc. ; 3095-v).

Assessing inhibitory activity for human HTRA2: 200 nM (final concentration) HTRA2 (R&D Systems, Inc.; 1458-HT) and 50 μM (final concentration) H2-Opt peptide substrate (Peptide Institute, Inc.).

Cross-reactivity with proteases other than HTRA1 was assessed using the cleavage of the peptide substrate as an index in the same manner as described in Example (3-2). Each HTRA1 inhibitory peptide did not inhibit the protease activity of any of the tested proteases at a final inhibitor concentration of 1 μM, indicating that the HTRA1 inhibitory peptide has an HTRA1-specific inhibitory effect (FIG. 5).

[0152] Example 4 Analysis of HTRA1 inhibitory peptide using X-ray crystallography

(4-1) Preparation of HTRA1 (catalyte)/HTRA1 inhibitory peptide complex

HTRA1 (catalyte) and an HTRA1 inhibitory peptide having the amino acid sequence shown in SEQ ID NO: 3 were each prepared according to the methods described in Examples (1-2) and (2-1). They were mixed under the following conditions: 20 mM Tris-HCl and 150 mM NaCl, pH 7.6. The complex was then isolated and purified by gel filtration (Superdex 200 10/300 GL).

(4-2) X-ray crystallography

получали анализом с использованием HKL2000. Фазу определяли методом молекулярной замены с использованием сериновой протеазы HTRA1 (ID PDB:3NZI) в качестве матрицы. После уточнения структуры определяли кристаллический комплекс HTRA1 (каталит) и пептида с разрешением 2,6

. Каждая из молекул HTRA1 и SPINK2 находилась в элементарной ячейке. В отношении молекулы SPINK2, то неполная молекулярная модель, содержащая сайт взаимодействия с HTRA1 (каталит), была сконструирована на основе информации о последовательности и наблюдаемой электронной плотности. Было подтверждено, что HTRA1-ингибирующий пептид связывается с областью, содержащей активный центр фермента HTRA1 (фиг. 6 и 7).The complex solution prepared in Example (4-1) was concentrated to 18 mg/ml and then mixed with a reservoir solution (1.0 M LiCl, 7.5% PEG6000 and 0.1 M Tris/HCl (pH 8.5) ) in a ratio of 1:1, and the mixture was crystallized by vapor diffusion. The resulting cubic single crystals were immersed in a reservoir solution containing 20% ethylene glycol and then frozen in liquid nitrogen. The frozen crystals were exposed to x-rays in a cryogenic air stream to obtain a diffraction image (photon factory BL5A: High Energy Energy Accelerator Research Organization). Scale data with a maximum resolution of 2.6

was obtained by analysis using HKL2000. The phase was determined by the molecular replacement method using HTRA1 serine protease (ID PDB:3NZI) as a template. After clarification of the structure, the crystal complex of HTRA1 (catalyte) and the peptide was determined with a resolution of 2.6

. Each of the HTRA1 and SPINK2 molecules was located in a unit cell. For the SPINK2 molecule, an incomplete molecular model containing the HTRA1 interaction site (catalyte) was constructed based on sequence information and observed electron density. The HTRA1 inhibitory peptide was confirmed to bind to the region containing the active site of the HTRA1 enzyme (FIGS. 6 and 7).

[0153]

Example 5 Retinal Protective Effects Conferred by HTRA1 Inhibition in a Rat Model of Light Induced Retinal Injury

(5-1) Obtaining a Rat Model of Light-Induced Retinal Damage

Light-induced rat retinal injury models represent models that induce retinal photoreceptor cell death upon exposure to light and are commonly used as animal models of retinal degeneration (Daniel T. Organisciak et al. (1996) Invest. Ophthalmol. Vis. Sci. Vol.37 (No.11): pp. 2243-2257). An ophthalmic solution consisting of 0.5% (w/v) tropicamide and 0.5% phenylephrine hydrochloride was instilled into the eye of dark adapted rats for 72 hours. The rats were then exposed to white light with illuminance of 5500 lux for 3 hours. The exposed rats were re-adapted to darkness for about 24 hours and then kept for 2 days under normal light-dark conditions. After euthanasia, eyeball samples were excised and fixed by immersion in a fixative consisting of picric acid 3.7% (w/v), formaldehyde 0.5-1% (w/v), methanol 0.2% (w/v). ./vol.), for 24 hours or more. After embedding in paraffin, thin sections were prepared. The sections were stained with hematoxylin-eosin, and the number of nuclei in the outer nuclear layer of the transverse section of the retina was determined to assess retinal damage. In a rat model of light-induced retinal injury, a marked decrease in the number of nuclei in the outer nuclear layer was observed as a result of light exposure.

(5-2) Confirmation of extracellular HTRA1 expression during retinal injury

In order to study the involvement of HTRA1 in light-induced retinal injury in a rat model, vitreous samples were taken from the model rats obtained in Example (5-1), and the expression level of HTRA1 was assessed by Western blotting. Vitreous body samples were subjected to SDS-PAGE under reducing conditions. Rat HTRA1 was detected using anti-human HTRA1/PRSS11 as primary antibody (R&D Systems, Inc.; AF2916) and horseradish peroxidase-conjugated sheep IgG as secondary antibody (R&D Systems, Inc.; HAF016) . An increased amount of HTRA1 in the vitreous was confirmed in the light-exposure group compared to the no-exposure group, suggesting that HTRA1 is involved in this model in light-induced retinal injury (Fig. 9).

(5-3) Retinal protective effect of HTRA1 inhibitory peptide in a rat model of light-induced retinal injury

Immediately prior to exposure to light, rats under anesthesia were injected intravitreally with 5 μl of HTRA1 inhibitory peptide H308 at a concentration of 0.04 mg/ml or 0.2 mg/ml. n=4 for the saline group and n=5 for other groups. Exposure to light resulted in a decrease in the number of nuclei in the outer nuclear layer on a cross-section of the retina in the normal saline group, while the effect of suppressing the decrease in the number of nuclei in the outer nuclear layer was confirmed in the HTRA1 inhibitory peptide group (Fig. 10). The results showed that the HTRA1 inhibitory peptide has a curative effect on tissue damage caused by HTRA1.

[0154] Example 6 Evaluation of an HTRA1 inhibitory peptide derivative

(6-1) Construction of pET 32a_HTRA1 inhibitory peptide H308_S16A_Kex2

An S16A derivative having an amino acid sequence in which 16Ser in the amino acid sequence shown in SEQ ID NO: 9 (FIG. 21) was replaced with Ala was prepared using the HTRA1 inhibitory peptide H308 as a template. Fragment C was amplified by PCR ((94°C for 15 s, 60°C for 30 s and 68°C for 15 s) × 30 cycles) using the following primers and KOD-plus - (Toyobo Co., Ltd.).

Primer 12: 5'-CCGCAGTTTGGTCTGTTTAGCAAATATCGTACCCCGAATTGT-3'

Primer 13: 5'-GCCATACCAGCATGGTCCGCACAATTCGGGGTACGATATTTGC-3'

Then fragment D was amplified by PCR ((94°C for 15 s, 60°C for 30 s and 68°C for 20 s) × 30 cycles) using the HTRA1 inhibitory peptide H308 as a template using the following primers and KOD-plus - (Toyobo Co., Ltd.).

Primer 14: 5'-GCGGACCATGCTGGTATGGCATGTGTTGCTCTGTATGAAC-3'

Primer 15: 5'-AAAACTCGAGTTAGCCGCCGCACGGACCATTGCGAATAA-3'

The desired DNA fragment was amplified by PCR ((94°C for 15 s, 60°C for 30 s and 68°C for 20 s) × 30 cycles) using fragments C and D, the following primers and KOD-plus - (Toyobo Co., Ltd.).

Primer 16: 5'-AAAAGGATCCCTGGACAAACGTGATCCGCAGTTTGGTCTGTTTAG-3'

Primer 15

The amplified fragment was subjected to agarose gel electrophoresis. Then, the desired DNA fragment was excised from the gel, and DNA was obtained using a QIAquick DNA gel isolation kit (Qiagen N.V.). The resulting DNA fragment and pET 32a (Novagen) were each treated with BamHI (New England BioLabs Inc.) and XhoI (New England BioLabs Inc.) at 37° C. for 1 hour or more. After agarose gel electrophoresis, the desired DNA fragments were excised from the gel and purified using the QIAquick PCR Purification Kit (Qiagen N.V.). The purified fragments were reacted overnight at 16°C for ligation using T4 DNA ligase (New England BioLabs Inc.). The ligation solution was added to E. coli JM109 (Toyobo Co., Ltd.) and the mixture was kept on ice for 30 minutes, then heat treated at 42°C for 45 seconds, then kept on ice for 5 minutes and inoculated into a plate with 2YT medium containing 0.1 mg/ml ampicillin, followed by overnight static culture at 37° C. to transform E. coli. Transformed E. coli was cultured and then miniprep and sequence analysis were performed to construct "pET 32a_HTRA1 inhibitory peptide H308_S16A_Kex2". The operation was performed according to the method described in Example (1-1-1).

(6-2) Preparation of expression vector HTRA1 inhibitory peptide_ N-terminal derivative

To obtain four derivatives of the N-terminal sequence (designated D1G, D1S, D1E and D1SLI respectively) of an HTRA1 inhibitory peptide having the amino acid sequence in which 1Asp in the amino acid sequence shown in SEQ ID NO: 9 (FIG. 21) was replaced with Gly , Ser, Glu or Ser-Leu-Ile, expression vectors were constructed in the same way as described in example (6-1). Four fragments of interest were each amplified by PCR ((94°C for 15 s, 60°C for 30 s and 68°C for 20 s) × 30 cycles) using fragments C and D, the following primers and KOD -plus - (Toyobo Co., Ltd.).

Primers for obtaining D1G

Primer 17: 5'-AAAAGGATCCCTGGACAAACGTGGCCCGCAGTTTGGTCTGTTTAG-3'

Primer 15

Primers for obtaining D1S

Primer 18: 5'-AAAAGGATCCCTGGACAAACGTAGCCCGCAGTTTGGTCTGTTTAG-3'

Primer 15

Primers for obtaining D1E

Primer 19: 5'-AAAAGGATCCCTGGACAAACGTGAACCGCAGTTTGGTCTGTTTAG-3'

Primer 15

Primers for obtaining D1SLI

Primer 20: 5'-AAAAGGATCCCTGGACAAACGTAGCCTGATTCCGCAGTTTGGTCTGTTTAG-3'

Primer 15

Each of the four amplified fragments was subjected to agarose gel electrophoresis. Then, the desired DNA fragment was excised from the gel, and DNA was obtained using a QIAquick DNA gel isolation kit (Qiagen N.V.). The resulting DNA fragment and pET 32a (Novagen) were each treated with BamHI (New England BioLabs Inc.) and XhoI (New England BioLabs Inc.) at 37° C. for 1 hour or more. After agarose gel electrophoresis, the desired DNA fragments were excised from the gel and purified using the QIAquick PCR Purification Kit (Qiagen N.V.). The purified fragments were reacted overnight at 16°C for ligation using T4 DNA ligase (New England BioLabs Inc.). The ligation solution was added to E. coli JM109 (Toyobo Co., Ltd.) and the mixture was kept on ice for 30 minutes, then heat treated at 42°C for 45 seconds, then kept on ice for 5 minutes and inoculated into a plate with 2YT medium containing 0.1 mg/ml ampicillin, followed by overnight static culture at 37° C. to transform E. coli. Трансформированную E.coli культивировали, и затем проводили минипреп и анализ последовательности для конструирования «pET 32a_HTRA1-ингибирующий пептид H308_D1G_S16A_Kex2», «pET 32a_HTRA1-ингибирующий пептид H308_D1S_S16A_Kex2», «pET 32a_HTRA1-ингибирующий пептид H308_D1E_S16A_Kex2» и «pET 32a_HTRA1-ингибирующий пептид H308_D1SL1_S16A_Kex2» . The operation was performed according to the method described in Example (1-1-1).

(6-3) Derivative of HTRA1 inhibitory peptide

E. coli Origami B (DE3) (Novagen) were transformed with each of the five vectors constructed in Examples (6-1) and (6-2) and cultured at 37°C using 2YT medium containing 0.1 mg/mL ampicillin. Then, IPTG (final concentration: 1 mM) was added to the mixture, and E. coli was cultured overnight at 16°C. The day after collection by centrifugation (3000 g, 20 min, 4°C), a lysate was prepared using BugBuster Master Mix (Novagen) and the His-tagged fusion protein of interest was purified using TALON metal chelate affinity resin ( Clontech Laboratories, Inc.). The thioredoxin tag and desired protein were then digested using Kex2 (indicated above) and purified using TALON. The resulting product was subjected to gel filtration (Superdex 75 10/300 GL) or reverse phase chromatography (YMC-Pack ODS-AM) to give five HTRA1 inhibitory peptide derivatives. The amino acid sequences of the derivatives are shown in SEQ ID NOs: 23-27 (FIGS. 35-39).

(6-4) Evaluation of the HTRA1 inhibitory peptide derivative

As a result of determining the activity of inhibition of HTRA1 (catalyst) in accordance with the method described in example (3-1), all derivatives had an inhibitory activity equivalent to that of H308 (figure 11).

[0155] Example 7 Evaluation of HTRA1 inhibitory peptide for binding activity to HTRA1 (catalyte)

Binding activity was evaluated by immunoprecipitation using three HTRA1 inhibitory peptides (H308, H321AT and H322AT) obtained in Example (1-2) and HTRA1 (catalyte) obtained in Example (2-1). 2.5 µg of each HTRA1 inhibitory peptide and 10 µg of HTRA1 (catalyte) were reacted at room temperature for 30 minutes. Then, 10 µl of TALON metal chelate affinity resin (Clontech Laboratories, Inc.) was added to the mixture. After an additional reaction of 30 minutes, the resin was isolated as an immunoprecipitation (IP) fraction and subjected to SDS-PAGE to assess binding activity. PBS was used as a buffer in the reaction.

[0156] When each of the three HTRA1 inhibitory peptides or HTRA1 (catalyst) reacted with TALON, an HTRA1 (catalyte) band fused to His tag only was detected in the input track. On the other hand, the band of each inhibitory peptide and enzyme was found only in the IP band where the inhibitory peptide reacted with HTRA1 (catalyte). Therefore, it was confirmed that each of the three HTRA1 inhibitory peptides binds to HTRA1 (catalyte).

[0157] Example 8 Evaluation of an HTRA1 inhibitory peptide for HTRA1 inhibitory activity

(8-1) Evaluation of an HTRA1 inhibitory peptide for HTRA1 inhibitory activity using a peptide substrate

The three HTRA1 inhibitory peptides (H308_D1G_S16A, H321AT_D1G_S16A and H322AT_D1G_S16A) constructed in Example 6 were evaluated for their HTRA1 (catalyte) or HTRA1 (total) inhibitory activity using the H2-Opt peptide substrate (n=3). Peptide substrate H2-Opt (Mca-IRRVSYSFK (Dnp) K) (Peptide Institute, Inc.: SEQ ID NO: 54, Figure 8) was dissolved at 10 mM in DMSO, diluted with assay buffer (50 mM Tris, 150 mM NaCl and 0.25% CHAPS, pH 8.0) and used at a final concentration of 10 μM. HTRA1 (HTRA1 (catalyte) or HTRA1 (total)); Example 2) and each HTRA1 inhibitory peptide diluted in assay buffer were mixed at 25 µl each and reacted at 37° C. for 20 minutes. Then, 50 μl of substrate diluted with assay buffer was added to the mixture. Fluorescent signal (excitation wave 328 nm/emission wave 393 nm) was measured using an Enspire (PerkinElmer, Inc.). The final concentration of HTRA1 was 100 nM and the final concentration of HTRA1 inhibitory peptide was 1.875 to 1000 nM. A black PROTEOSAVE® R9696 plate (Sumitomo Bakelite Co., Ltd.) was used in the reaction and measurement.

[0158] The rate of degradation of the peptide substrate in the presence of the HTRA1 inhibitory peptide at each concentration was calculated. When the cleavage rate at 0 nM inhibitor concentration was taken as 100%, the HTRA1 (catalyte) and HTRA1 (total) inhibition activity of each HTRA1 inhibitory peptide was evaluated.

As a result of calculating the 50% inhibitory concentration (IC ₅₀ ) using GraphPad Prism (version 5.0; GraphPad Software Inc.), it was found that all HTRA1 inhibitory peptides inhibit the activity of the HTRA1 enzyme (catalyte) and HTRA1 (total) at a low concentration (Fig. .66).

[0159] HTRA1 inhibitory activity of HTRA1 inhibitory peptide

[0160]

table 2 IC50 (nM) for HTRA1 (catalyte) IC50 (nM) for HTRA1 (full) H308_D1G_S16A 7.9±1.3 9.1±1.4 H321AT_D1G_S16A 9.0±0.6 12.0±1.9 H322AT_D1G_S16A 12.9±0.2 12.2±2.2

[0161] (8-2) Evaluation of an HTRA1 inhibitory peptide for HTRA1 inhibitory activity using a protein substrate

The HTRA1 inhibitory activity of the HTRA1 inhibitory peptide was evaluated with human vitronectin as a protein substrate. The operation was carried out as described in Example (3-2).

As in Example (8-1), the HTRA1 inhibitory peptides also clearly showed inhibition of HTRA1 (catalyte) when human vitronectin was used as a substrate (FIG. 67).

[0162] (8-3) Evaluation of HTRA1 inhibitory peptide for specificity

Specificity for other proteases was evaluated using cleavage of the peptide substrate as an index. Operations for bovine trypsin, bovine β-chymotrypsin, protein C, tryptase, chymase, thrombin, plasmin, tPA, plasma kallikrein, matriptase, uPA and HTRA2 were performed according to the method described in example (3-3) (n=3). Procedures for measuring inhibitory activity for other proteases and combinations of protease and substrate were as follows.

[0163] A black ^PROTEOSAVE® R9696 plate (Sumitomo Bakelite Co., Ltd.) was used in the reaction and measurement. Each protease and each sample (final concentration: 1 μM) diluted with assay buffer was mixed at 25 μl each and reacted at 37° C. for 20 minutes. Then, 50 µl of each substrate diluted with assay buffer was added to the mixture. Fluorescent signal was measured using Enspire (PerkinElmer, Inc.).

[0164] Evaluation of inhibitory activity for human trypsin: 1 nM (final concentration) trypsin (Sigma-Aldrich Co. LLC; T6424) and 100 μM (final concentration) Boc-VPR-AMC peptide substrate, fluorogenic peptide substrate (R & D Systems , Inc.; ES011), fluorescent signal: 380 nm excitation wave/460 nm emission wave.

[0165] Evaluation of inhibitory activity for human chymotrypsin: 10 nM (final concentration) chymotrypsin (Sigma-Aldrich Co. LLC; C8946) and 10 μM (final concentration) Suc-Leu-Leu-Val-Tyr-MCA peptide substrate (Peptide Institute , Inc.; 3120-v), fluorescent signal: 380 nm excitation wave/460 nm emission wave.

[0166] Evaluation of inhibitory activity for human factor XIIa: 100 nM (final concentration) factor alpha-XIIa (Enzyme Research Laboratories Inc.) and 100 μM (final concentration) Pyr-Gly-Arg-MCA peptide substrate (Peptide Institute, Inc. ; 3145-v), fluorescent signal: 380 nm excitation wave/460 nm emission wave.

[0167] Evaluation of inhibitory activity for human MMP-2: 1 nM (final) tryptase (Calbiochem; PF023) and 100 μM (final) peptide substrate MOCAx-KPLGL-A2pr (Dnp)-AR (Peptide Institute, Inc.; 3226-v), fluorescent signal: 328 nm excitation wave/393 nm emission wave.

[0168] Evaluation of inhibitory activity for human TPP1: 0.5 μg/ml (final concentration) TPP1 (Calbiochem; 2237-SE) and 200 μM (final concentration) AAF-MCA peptide substrate (Peptide Institute, Inc.; 3201-v ), fluorescent signal: excitation wave 380 nm/emission wave 460 nm.

[0169] Cross-reactivity with proteases other than HTRA1 was assessed using cleavage of the peptide substrate as an index. Each HTRA1 inhibitory peptide did not inhibit the protease activity of any of the proteases at a final concentration of 1 μM, indicating that the HTRA1 inhibitory peptide has an HTRA1-specific inhibitory effect (FIG. 68).

[0170] Example 9 Evaluation of HTRA1 inhibitory peptide for binding activity to HTRA1 (catalyte)

Binding activity was assessed by immunoprecipitation according to the procedure described in Example 7 using the three HTRA1 inhibitory peptides obtained in Example 6 and HTRA1 (catalyte) obtained in Example (2-1).

When each of the three HTRA1 inhibitory peptides or HTRA1 (catalyte) was reacted with TALON, only the HTRA1 (catalyte) band fused to the His tag was detected in the input track. On the other hand, the band of each inhibitory peptide and enzyme was detected only in the IP band where the inhibitory peptide reacted with HTRA1 (catalyte). Therefore, each of the three HTRA1 inhibitory peptides was confirmed to bind to HTRA1 (catalyte) (FIG. 69).

[0171] Example 10 Retinal Protective Effects Conferred by HTRA1 Inhibition in a Rat Model of Light Induced Retinal Damage (Part 2)

The retinal protective effect of the three HTRA1 inhibitory peptides obtained in Example 6 was evaluated using the rat model of light-induced retinal injury obtained in Example (5-1). The operation was carried out following example 5. n=6 for all groups.

The results of the pathological evaluation of the retina are shown in FIG. 70. Three HTRA1 inhibitory peptides showed a pronounced inhibitory effect on light-induced reduction in the number of nuclei in the outer nuclear layer.

[0172] Example 11 Protective effect on retinal pigment epithelial cells by inhibiting HTRA1 in a rabbit model of retinal injury induced by feeding a high-fat hydroquinone diet

(11-1) Establishing a model of retinal injury in rabbits by feeding a high-fat diet containing hydroquinone

The model of retinal injury obtained using a high fat diet (HFD) and the presence of hydroquinone (HQ) represents a model in which oxidative stress is induced by a pro-oxidant causing damage to the retina. This model has only been described in mice (Diego G. Espinosa-Heidmann et al. (2006) Invest. Ophthalmol. Vis. Sci., Vol. 47 (No. 2): p. 729-737). Therefore, 3-year-old JW rabbits were fed an RC4 diet (Oriental Yeast Co., Ltd.) containing 1.5% (w/v) coconut oil - 0.25% (w/v) for 4 months. cholesterol - 1.5% (w/v) peanut butter - 2.4% (w/v)) hydroquinone (HFD-HQ) to obtain a model of retinal damage in rabbits. After euthanasia, the eyeball samples were removed and the anterior segment of the eye was removed by an external incision at a distance of approximately 5 mm from the corneal limbus. Then the vitreous body was additionally separated. The retina-choroid-sclera was then fixed by immersion in 4% (w/v) paraformaldehyde fixative for 24 hours or more. After fixation, the choroid was separated and immunostained using the monoclonal antibody ZO-1 as primary antibody (ZO1-1A12) (Thermo Fisher Scientific Inc.; 33-9100) and chicken anti-mouse IgG (H+L) cross-adsorbed secondary antibody conjugated with Alexa Fluor 594 as secondary antibody (Thermo Fisher Scientific Inc.; A-21201). The stained choroid was examined under a fluorescence microscope (BZ-9000; Keyence Corp.). The area of stained retinal pigment epithelial (RPE) cells was determined to assess damage to RPE cells.

In FIG. 71 shows stained images of RPE cells of a 12-week-old rabbit, a 3-year-old rabbit, and a 3-year-old rabbit fed an HFD-HQ diet (FIG. 71(A)) and a plot of mean RPE cell areas (FIG. 71(B)). It was confirmed that the RPE cells were more hypertrophied in the 3 year old rabbit than in the 12 week old rabbit, and they underwent additional hypertrophy as a result of feeding the HFD-HQ diet. It was confirmed that damage appeared in RPE cells. Similar changes have been observed in the eyeballs of patients with age-related macular degeneration (Ding J.D. et al. (2011) Proc. Natl. Acad. Sci. U S A., Vol. 108 (No. 28): p. 279-87).

[0173] (11-2) Increased expression of AMD-associated factor C3 during retinal injury

To assess the expression of AMD-associated factor, tissue samples were taken from the retina and RPE/choroid, respectively, in a rabbit model of retinal injury. mRNA was extracted using the RNeasy mini kit (Qiagen NV) and then subjected to a reverse transcription reaction using ^TaqMan® Gene Expression Master Mix (Thermo Fisher Scientific Inc.). mRNA levels of complement component 3 (C3) and internal standard β-actin were quantified using the ^TaqMan® Gene Expression Assay (Oc03397832_g1 and Oc03824857_g1; Thermo Fisher Scientific Inc.) using the 7900HT Fast Real-Time PCR System. (Applied Biosystems, Inc.). The analysis was performed at n=4 for 3 year old rabbits and n=10 for 3 year old rabbits fed the HFD-HQ diet.

[0174] C3 expression levels in the retina and in RPE cells and choroid are shown in FIG. 71(C) and (D). For both tissues, it was confirmed that the expression level of C3 was increased in the group of rabbits fed the HFD-HQ diet.

[0175] (11-3) Increase in HTRA1 protein during retinal injury

For a rabbit model study of HTRA1 involvement in retinal injury, vitreous samples were taken from the model rabbits obtained in Example (11-1) and enzymatically digested with Trypsin/Lys-C (Promega Corp.). The HTRA1 peptide fragment was then quantified using LC (EASY-nLC 1000; Thermo Fisher Scientific Inc.)-MS (TripleTOF 6600; AB Sciex Pte. Ltd). The level of HTRA1 protein was found to be elevated in the vitreous of rabbits fed the HFD-HQ diet (FIG. 71(E)). Based on these results, RPE cell hypertrophy and increased expression of AMD-associated factor C3 and HTRA1 were confirmed, indicating that the rabbit model of retinal injury is useful in studies of age-related retinal diseases.

[0176] (11-4) Retinal protective effect of an HTRA1 inhibitor in a rabbit model of retinal injury

The retinal protective effect of the HTRA1 inhibitory peptide H308 prepared in Example 1 was evaluated using a rabbit model. 2 months after starting the HFD-HQ diet, 50 µl of a 40 mg/ml H308 solution was injected intravitreally into one eye under anesthesia. Normal saline was injected intravitreally into the opposite eye. n=5 for all groups.

4 months after the start of feeding, the hypertrophy of RPE cells in model animals was assessed. The results are shown in FIG. 72. The HTRA1 inhibitor had an inhibitory effect on RPE cell hypertrophy as seen from both measures, ie. the average area of RPE cells (Fig. 72 (A)) and the number of hypertrophied RPE cells with a cell area of 1500 μm ² or higher (Fig. 72 (B)). As shown in FIG. 71 (E), an increase in HTRA1 was confirmed in vitreous models suggesting the involvement of HTRA1 in RPE cell damage resulting from feeding the HFD-HQ diet. Thus, an HTRA1 inhibitory peptide is useful as an anti-ageing macular degeneration agent. The results of this test showed that the HTRA1 inhibitory peptide is useful in preventing, in particular, the dry form of age-related macular degeneration.

The presence of the HTRA1 inhibitory peptide was confirmed in the retina of a normal rabbit treated with the HTRA1 inhibitory peptide, indicating high tissue penetration of the HTRA1 inhibitory peptide.

[0177] Example 12 Inhibitory Effect of HTRA1 Inhibitory Peptide in VEGF mRNA Induction Assay Using ARPE-19 Human Retinal Pigment Epithelial Cells

ARPE-19 cells were cultured to confluence in 12 mm Transwell culture inserts with 0.4 μm sterile polyester membrane (Corning Inc.) at 37°C and 5% CO ₂ using DMEM/F-12 medium (Wako Pure Chemical Industries, Ltd.) containing 10% fetal bovine serum (FBS) and penicillin-streptomycin mixture (Thermo Fisher Scientific Inc.). The cells were then cultured in DMEM/F-12 without FBS for 5 days. H ₂ O ₂ (final concentration: 500 μM) was added to the top and bottom layers of the chamber, and normal human serum complement (Quidel Corp.) (final concentration: 25%) was added to the top layer of the chamber. Each of HTRA1 (Example 2-2), inactive HTRA1 protease, mutant HTRA1 (S328A) (Example 2-3), or HTRA1 inhibitory peptide H308_D1G_S16A (Example 6) was additionally added at a final concentration of 1 μM to the top and bottom layers of the chamber. After four hours, the culture supernatant was removed and the cells were washed with PBS. Then, mRNA was extracted using RT SuperPrep™ cell lysis kit for qPCR (Toyobo Co., Ltd.) and subjected to a reverse transcription reaction. VEGF mRNA levels were quantified using the ^TaqMan® Gene Expression Assay (Hs000900055_m1 and Hs02786624_g1; Thermo Fisher Scientific Inc.) using the 7900HT Fast Real-Time PCR System (Applied Biosystems, Inc.). GAPDH was used to normalize mRNA levels.

The results are shown in FIG. 74. H ₂ O ₂ , normal human serum complement and HTRA1 supplementation markedly increased VEGF mRNA levels compared to conditions involving H ₂ O ₂ supplementation, normal human serum complement and inactive HTRA1, HTRA1 mutant (S328A). It was confirmed that the co-addition of the HTRA1 inhibitory peptide H308_D1G_S16A suppresses VEGF expression. Induction of VEGF in retinal pigment epithelial cells has been reported to be important in the pathogenesis of wet age-related macular degeneration (Klettner A. et al. (2009) Graefes Arch. Clin. Exp. Ophthalmol., Vol. 247: p. 1487-1492). In addition, it is believed that such abnormal VEGF induction is involved not only in pathogenesis, but also in the maintenance of the pathological state. Thus, administration of a peptide of the present invention, such as the HTRA1 inhibitory peptide H308_D1G_S16A, is effective in preventing and treating wet age-related macular degeneration.

[0178] Example 13 Inhibitory effect of HTRA1 inhibitory peptide H308_D1G_S16A on human umbilical vein endothelial cell (HUVEC) migration

(13-1) HUVEC Migration Test

HUVEC (Kurabo Industries Ltd.) were cultured for 18 h at 37°C and 5% CO ₂ in medium (0.1% BSA-containing serum-free EGM) in which EBM(TM)-2 basal medium (Lonza Walkersville, Inc.) containing 0.1% BSA was fortified with the EGM(TM)-2 SingleQuots(TM) additive factor kit, excluding serum and VEGF. The cells were then brought to a titer of 4×10 ⁵ cells/ml with 0.1% BSA-containing serum-free EGM medium. 4x10 ⁵ cells/ml of HUVEC suspension was added at 50 μl/well to the top layer of the chamber of a 96-well Multiwell Permeable Support system with a 3.0 μm high density PET membrane (Corning Inc.) with a gelatin-coated membrane. Then each sample described below (medium 1, 2 or 3) was added at the rate of 210 μl/well to the bottom layer of the chamber (n=3). 0.1% BSA-containing serum-free EGM medium was added at 50 µl/well to the top layer of the chamber without adding HUVEC, and 0.1% BSA-containing serum-free EGM medium was added at 210 µl/well to the bottom layer of the chamber (n= 3).

Medium 1: 0.1% BSA-containing serum-free EGM medium

Medium 2: EBM(TM)-2 Enriched with All Additive Factor EGM(TM)-2 SingleQuots(TM) (EGM Growth Medium)

Medium 3: EGM growth medium containing 300 nM H308_D1G_S16A.

A 96-well FluoroBlok HTS Multiwell Permeable Support system with cell addition and sample was incubated for 2 hours at 37°C and 5% CO ₂ . HUVEC migrating to the bottom layer were washed with PBS and then stained for 15 min with 0.1% BSA-containing serum-free EGM medium containing 4 μg/ml calcein AM (Thermo Fisher Scientific Inc.). The medium was then replaced with PBS. Fluorescence intensity (excitation wavelength/emission wavelength: 485 nm/535 nm) of each well was measured using a plate reader (ARVO-MX, PerkinElmer, Inc.), and migrating cells were counted for each well using the following equation:

migrated cells=average fluorescence intensity of wells containing HUVEC (n=3) - average fluorescence intensity of empty wells (n=3).

The results are shown in FIG. 75. The HTRA1 inhibitory peptide H308_D1G_S16A was confirmed to have an inhibitory effect on HUVEC migration induced in serum-containing medium. Therefore, it was found that the peptide of the present invention exhibits an inhibitory effect on angiogenesis, which is a feature of the wet form of age-related macular degeneration.

[0179] Example 14 Retinal Protective Effect of HTRA1 Inhibitory Peptide in Rabbit Model of Retinal Injury (Part 2)

The HTRA1 inhibitory peptide H308 prepared in Example 1 or one of the three HTRA1 inhibitory peptides prepared in Example 6 was evaluated for their therapeutic effect on retinal injury using the rabbit retinal injury model prepared and evaluated in Examples (11-1 )-(11-3). 50 μl of a 40 mg/ml inhibitory peptide solution was injected intravitreally into one eye of each model animal under anesthesia, and the animals were kept for 2 months. Normal saline was injected intravitreally into the opposite eye. n=5 for all groups.

An increase in the area of RPE cells or an increase in the number of RPE cells was found in the saline group, while an increase in the area of RPE cells or an increase in the number of RPE cells was suppressed in the HTRA1 inhibitory peptide group. Thus, it can be confirmed that the HTRA1 inhibitory peptide can be used as an anti-ageing macular degeneration agent. In this example, it can be confirmed that the HTRA1 inhibitory peptide is useful in the treatment of dry age-related macular degeneration in particular.

Industrial Applicability

[0180] The peptide provided by the present invention and the pharmaceutical composition containing the peptide are suitable for treatment or prophylaxis, etc. age-related macular degeneration and the like.

Free text sequence listing

[0181]

SEQ ID NO: 1 - Amino acid sequence of human SPINK2 (Fig. 13)

SEQ ID NO: 2 - Nucleotide sequence encoding human SPINK2 amino acid sequence (Fig. 14)

SEQ ID NO: 3 - Amino acid sequence of H218 peptide (Fig. 15)

SEQ ID NO: 4 - Nucleotide sequence encoding the amino acid sequence of the H218 peptide (Fig. 16)

SEQ ID NO: 5 - Amino acid sequence of H223 peptide (Fig. 17)

SEQ ID NO: 6 - Nucleotide sequence encoding the amino acid sequence of the H223 peptide (Fig. 18)

SEQ ID NO: 7 - Amino acid sequence of H228 peptide (Fig. 19)

SEQ ID NO: 8 - Nucleotide sequence encoding the amino acid sequence of the H228 peptide (Fig. 20)

SEQ ID NO: 9 - Amino acid sequence of H308 peptide (Fig. 21)

SEQ ID NO: 10 - Nucleotide sequence encoding the amino acid sequence of the H308 peptide (Fig. 22)

SEQ ID NO: 11 - Amino acid sequence of H321 peptide (Fig. 23)

SEQ ID NO: 12 - Nucleotide sequence encoding the amino acid sequence of the H321 peptide (Fig. 24)

SEQ ID NO: 13 - Amino acid sequence of H322 peptide (Fig. 25)

SEQ ID NO: 14 - Nucleotide sequence encoding the amino acid sequence of the H322 peptide (Figure 26)

SEQ ID NO: 15 - Amino acid sequence of the peptide derivative H308AT (Fig. 27)

SEQ ID NO: 16 - Nucleotide sequence encoding the amino acid sequence of the H308AT peptide derivative (Fig. 28)

SEQ ID NO: 17 - Amino acid sequence of peptide derivative H321AT (Fig. 29)

SEQ ID NO: 18 - Nucleotide sequence encoding the amino acid sequence of the H321AT peptide derivative (Fig. 30)

SEQ ID NO: 19 - Amino acid sequence of peptide derivative H322AT (Fig. 31)

SEQ ID NO: 20 - Nucleotide sequence encoding the amino acid sequence of the H322AT peptide derivative (Fig. 32)

SEQ ID NO: 21 - Amino acid sequence of M7 peptide (Fig. 33)

SEQ ID NO: 22 - nucleotide sequence encoding the amino acid sequence of the M7 peptide (Fig. 34)

SEQ ID NO: 23 - Amino acid sequence of peptide derivative H308_S16A (Fig. 35)

SEQ ID NO: 24 - Amino acid sequence of the peptide derivative H308_D1G_S16A (Fig. 36)

SEQ ID NO: 25 - Amino acid sequence of the peptide derivative H308_D1S_S16A (figure 37)

SEQ ID NO: 26 - Amino acid sequence of the peptide derivative H308_D1E_S16A (Fig. 38)

SEQ ID NO: 27 - Amino acid sequence of the peptide derivative H308_D1SLI_S16A (Fig. 39)

SEQ ID NO: 28 - Amino acid sequence of the peptide derivative H321AT_D1G_S16A (Fig. 40)

SEQ ID NO: 29 - Amino acid sequence of the peptide derivative H322AT_D1G_S16A (Fig. 41)

SEQ ID NO: 30 - General formula of HTRA1 inhibitory peptide (Fig. 42)

SEQ ID NO: 31 - Amino acid sequence consisting of S-tag and linker (Fig. 43)

SEQ ID NO: 32 - Amino acid sequence of the C-terminal hexamer (Fig. 44)

SEQ ID NO: 33 - Nucleotide sequence of primer 1 (Fig. 45)

SEQ ID NO: 34 - Nucleotide sequence of primer 2 (Fig. 46)

SEQ ID NO: 35 - Nucleotide sequence of primer 3 (Fig. 47)

SEQ ID NO: 36 - Nucleotide sequence of primer 4 (Fig. 48)

SEQ ID NO: 37 - Nucleotide sequence of primer 5 (Fig. 49)

SEQ ID NO: 38 - Nucleotide sequence of primer 6 (Figure 50)

SEQ ID NO: 39 - Nucleotide sequence of primer 7 (Fig. 51)

SEQ ID NO: 40 - Nucleotide sequence of primer 8 (Figure 52)

SEQ ID NO: 41 - Nucleotide sequence of primer 9 (Fig. 53)

SEQ ID NO: 42 - Nucleotide sequence of primer 10 (Figure 54)

SEQ ID NO: 43 - Nucleotide sequence of primer 11 (Fig. 55)

SEQ ID NO: 44 - Nucleotide sequence of primer 12 (Fig. 56)

SEQ ID NO: 45 - Nucleotide sequence of primer 13 (Fig. 57)

SEQ ID NO: 46 - Nucleotide sequence of primer 14 (Fig. 58)

SEQ ID NO: 47 - Nucleotide sequence of primer 15 (Fig. 59)

SEQ ID NO: 48 - Nucleotide sequence of primer 16 (Fig. 60)

SEQ ID NO: 49 - Nucleotide sequence of primer 17 (Fig. 61)

SEQ ID NO: 50 - Nucleotide sequence of primer 18 (Fig. 62)

SEQ ID NO: 51 - Nucleotide sequence of primer 19 (Fig. 63)

SEQ ID NO: 52 - Nucleotide sequence of primer 20 (Fig. 64)

SEQ ID NO: 53 - Amino acid sequence of human HTRA1 (complete) (Fig. 65)

SEQ ID NO: 54 - Amino acid sequence H2-Opt (Fig. 8)

SEQ ID NO: 55 - Nucleotide sequence of primer 21 (Fig. 76)

SEQ ID NO: 56 - Nucleotide sequence of primer 22 (Fig. 77)

--->

Sequence list

<110> DAIICHI SANKYO COMPANY, LIMITED

<120> Peptide for the treatment of age-related macular degeneration

<130>FP1726

<150>JP2016-249020

<151> 2016-12-22

<160> 56

<170> Patent In version 3.5

<210> 1

<211> 63

<212> PROTEIN

<213> Homo sapiens

<400> 1

Asp Pro Gln Phe Gly Leu Phe Ser Lys Tyr Arg Thr Pro Asn Cys Ser

1 5 10 15

Gln Tyr Arg Leu Pro Gly Cys Pro Arg His Phe Asn Pro Val Cys Gly

20 25 30

Ser Asp Met Ser Thr Tyr Ala Asn Glu Cys Thr Leu Cys Met Lys Ile

35 40 45

Arg Glu Gly Gly His Asn Ile Lys Ile Ile Arg Asn Gly Pro Cys

50 55 60

<210> 2

<211> 192

<212> DNA

<213> Homo sapiens

<400> 2

gatccgcagt ttggtctgtt tagcaaatat cgtaccccga attgtagcca gtatcgtctg 60

cctggttgtc cgcgtcattt taatccggtt tgtggtagcg atatgagcac ctatgcaaat 120

gaatgtaccc tgtgcatgaa aattcgtgaa ggtggccata atattaaaat tattcgcaat 180

ggtccgtgct aa 192

<210> 3

<211> 65

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial

<400> 3

Asp Pro Gln Phe Gly Leu Phe Ser Lys Tyr Arg Thr Pro Asn Cys Leu

1 5 10 15

Lys Ser Glu Gly Met Ala Cys Tyr Ala Tyr Tyr Glu Pro Val Cys Gly

20 25 30

Ser Asp Met Ser Thr Tyr Ala Asn Glu Cys Thr Leu Cys Met Lys Ile

35 40 45

Arg Glu Gly Gly His Asn Ile Lys Ile Ile Arg Asn Gly Pro Cys Gly

50 55 60

gly

65

<210> 4

<211> 198

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial

<400> 4

gatccgcagt ttggtctgtt tagcaaatat cgtaccccga attgtctgaa atctgaaggt 60

atggcttgtt acgcttacta cgaaccggtt tgtggtagcg atatgagcac ctatgcaaat 120

gaatgtaccc tgtgcatgaa aattcgtgaa ggtggccata atattaaaat tattcgcaat 180

ggtccgtgcg gcggctaa 198

<210> 5

<211> 65

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial

<400> 5

Asp Pro Gln Phe Gly Leu Phe Ser Lys Tyr Arg Thr Pro Asn Cys Thr

1 5 10 15

Met Asp Met Gly Met Ala Cys Trp Ala Phe Tyr Glu Pro Val Cys Gly

20 25 30

Ser Asp Met Ser Thr Tyr Ala Asn Glu Cys Thr Leu Cys Met Lys Ile

35 40 45

Arg Glu Gly Gly His Asn Ile Lys Ile Ile Arg Asn Gly Pro Cys Gly

50 55 60

gly

65

<210> 6

<211> 198

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial

<400> 6

gatccgcagt ttggtctgtt tagcaaatat cgtaccccga attgtactat ggacatgggt 60

atggcttgtt gggctttcta cgaaccggtt tgtggtagcg atatgagcac ctatgcaaat 120

gaatgtaccc tgtgcatgaa aattcgtgaa ggtggccata atattaaaat tattcgcaat 180

ggtccgtgcg gcggctaa 198

<210> 7

<211> 65

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial

<400> 7

Asp Pro Gln Phe Gly Leu Phe Ser Lys Tyr Arg Thr Pro Asn Cys Gly

1 5 10 15

His Tyr Asn Gly Trp Ala Cys Gln Ala Phe Phe Glu Pro Val Cys Gly

20 25 30

Ser Asp Met Ser Thr Tyr Ala Asn Glu Cys Thr Leu Cys Met Lys Ile

35 40 45

Arg Glu Gly Gly His Asn Ile Lys Ile Ile Arg Asn Gly Pro Cys Gly

50 55 60

gly

65

<210> 8

<211> 198

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial

<400> 8

gatccgcagt ttggtctgtt tagcaaatat cgtaccccga attgtggtca ttacaacggt 60

tgggcttgtc aggctttctt cgaaccggtt tgtggtagcg atatgagcac ctatgcaaat 120

gaatgtaccc tgtgcatgaa aattcgtgaa ggtggccata atattaaaat tattcgcaat 180

ggtccgtgcg gcggctaa 198

<210> 9

<211> 65

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial

<400> 9

Asp Pro Gln Phe Gly Leu Phe Ser Lys Tyr Arg Thr Pro Asn Cys Ser

1 5 10 15

Asp His Ala Gly Met Ala Cys Val Ala Leu Tyr Glu Pro Val Cys Gly

20 25 30

Ser Asp Met Ser Thr Tyr Glu Asn Glu Cys Val Leu Cys Met Lys Ile

35 40 45

Arg Glu Gly Gly His Asn Ile Lys Ile Ile Arg Asn Gly Pro Cys Gly

50 55 60

gly

65

<210> 10

<211> 198

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial

<400> 10

gatccgcagt ttggtctgtt tagcaaatat cgtaccccga attgtagcga ccatgctggt 60

atggcatgtg ttgctctgta tgaaccggtt tgtggtagcg atatgagcac ctatgaaaat 120

gaatgtgttc tgtgcatgaa aattcgtgaa ggtggccata atattaaaat tattcgcaat 180

ggtccgtgcg gcggctaa 198

<210> 11

<211> 65

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial

<400> 11

Asp Pro Gln Phe Gly Leu Phe Ser Lys Tyr Arg Thr Pro Asn Cys Ser

1 5 10 15

Asp Phe Asp Gly Met Ala Cys Tyr Ala Phe Tyr Glu Pro Val Cys Gly

20 25 30

Ser Asp Met Ser Thr Tyr Met Asn Glu Cys Ala Leu Cys Met Lys Ile

35 40 45

Arg Glu Gly Gly His Asn Ile Lys Ile Ile Arg Asn Gly Pro Cys Gly

50 55 60

gly

65

<210> 12

<211> 198

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial

<400> 12

gatccgcagt ttggtctgtt tagcaaatat cgtaccccga attgtagcga cttcgacggt 60

atggcatgtt acgctttcta tgaaccggtt tgtggtagcg atatgagcac ctatatgaat 120

gaatgtgctc tgtgcatgaa aattcgtgaa ggtggccata atattaaaat tattcgcaat 180

ggtccgtgcg gcggctaa 198

<210> 13

<211> 65

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial

<400> 13

Asp Pro Gln Phe Gly Leu Phe Ser Lys Tyr Arg Thr Pro Asn Cys Ser

1 5 10 15

Gln His Glu Gly Met Ala Cys Tyr Ala Leu Tyr Glu Pro Val Cys Gly

20 25 30

Ser Asp Met Ser Thr Tyr Val Asn Glu Cys Ala Leu Cys Met Lys Ile

35 40 45

Arg Glu Gly Gly His Asn Ile Lys Ile Ile Arg Asn Gly Pro Cys Gly

50 55 60

gly

65

<210> 14

<211> 198

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial

<400> 14

gatccgcagt ttggtctgtt tagcaaatat cgtaccccga attgtagcca gcatgaaggt 60

atggcatgtt acgctctgta tgaaccggtt tgtggtagcg atatgagcac ctatgttaat 120

gaatgtgctc tgtgcatgaa aattcgtgaa ggtggccata atattaaaat tattcgcaat 180

ggtccgtgcg gcggctaa 198

<210> 15

<211> 65

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial

<400> 15

Asp Pro Gln Phe Gly Leu Phe Ser Lys Tyr Arg Thr Pro Asn Cys Ser

1 5 10 15

Asp His Ala Gly Met Ala Cys Val Ala Leu Tyr Glu Pro Val Cys Gly

20 25 30

Ser Asp Met Ser Thr Tyr Ala Asn Glu Cys Thr Leu Cys Met Lys Ile

35 40 45

Arg Glu Gly Gly His Asn Ile Lys Ile Ile Arg Asn Gly Pro Cys Gly

50 55 60

gly

65

<210> 16

<211> 198

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial

<400> 16

gatccgcagt ttggtctgtt tagcaaatat cgtaccccga attgtagcga ccatgctggt 60

atggcatgtg ttgctctgta tgaaccggtt tgtggtagcg atatgagcac ctatgcaaat 120

gaatgtaccc tgtgcatgaa aattcgtgaa ggtggccata atattaaaat tattcgcaat 180

ggtccgtgcg gcggctaa 198

<210> 17

<211> 65

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial

<400> 17

Asp Pro Gln Phe Gly Leu Phe Ser Lys Tyr Arg Thr Pro Asn Cys Ser

1 5 10 15

Asp Phe Asp Gly Met Ala Cys Tyr Ala Phe Tyr Glu Pro Val Cys Gly

20 25 30

Ser Asp Met Ser Thr Tyr Ala Asn Glu Cys Thr Leu Cys Met Lys Ile

35 40 45

Arg Glu Gly Gly His Asn Ile Lys Ile Ile Arg Asn Gly Pro Cys Gly

50 55 60

gly

65

<210> 18

<211> 198

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial

<400> 18

gatccgcagt ttggtctgtt tagcaaatat cgtaccccga attgtagcga cttcgacggt 60

atggcatgtt acgctttcta tgaaccggtt tgtggtagcg atatgagcac ctatgcaaat 120

gaatgtaccc tgtgcatgaa aattcgtgaa ggtggccata atattaaaat tattcgcaat 180

ggtccgtgcg gcggctaa 198

<210> 19

<211> 65

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial

<400> 19

Asp Pro Gln Phe Gly Leu Phe Ser Lys Tyr Arg Thr Pro Asn Cys Ser

1 5 10 15

Gln His Glu Gly Met Ala Cys Tyr Ala Leu Tyr Glu Pro Val Cys Gly

20 25 30

Ser Asp Met Ser Thr Tyr Ala Asn Glu Cys Thr Leu Cys Met Lys Ile

35 40 45

Arg Glu Gly Gly His Asn Ile Lys Ile Ile Arg Asn Gly Pro Cys Gly

50 55 60

gly

65

<210> 20

<211> 198

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial

<400> 20

gatccgcagt ttggtctgtt tagcaaatat cgtaccccga attgtagcca gcatgaaggt 60

atggcatgtt acgctctgta tgaaccggtt tgtggtagcg atatgagcac ctatgcaaat 120

gaatgtaccc tgtgcatgaa aattcgtgaa ggtggccata atattaaaat tattcgcaat 180

ggtccgtgcg gcggctaa 198

<210> 21

<211> 65

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial

<400> 21

Asp Pro Gln Phe Gly Leu Phe Ser Lys Tyr Arg Thr Pro Asn Cys Ser

1 5 10 15

Asp His Ala Gly Met Ala Cys Val Ala Phe Tyr Glu Pro Val Cys Gly

20 25 30

Ser Asp Met Ser Thr Tyr Ala Asn Glu Cys Thr Leu Cys Met Lys Ile

35 40 45

Arg Glu Gly Gly His Asn Ile Lys Ile Ile Arg Asn Gly Pro Cys Gly

50 55 60

gly

65

<210> 22

<211> 198

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial

<400> 22

gatccgcagt ttggtctgtt tagcaaatat cgtaccccga attgtagcga ccatgctggt 60

atggcatgtg ttgcttttta tgaaccggtt tgtggtagcg atatgagcac ctatgcaaat 120

gaatgtaccc tgtgcatgaa aattcgtgaa ggtggccata atattaaaat tattcgcaat 180

ggtccgtgcg gcggctaa 198

<210> 23

<211> 65

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial

<400> 23

Asp Pro Gln Phe Gly Leu Phe Ser Lys Tyr Arg Thr Pro Asn Cys Ala

1 5 10 15

Asp His Ala Gly Met Ala Cys Val Ala Leu Tyr Glu Pro Val Cys Gly

20 25 30

Ser Asp Met Ser Thr Tyr Glu Asn Glu Cys Val Leu Cys Met Lys Ile

35 40 45

Arg Glu Gly Gly His Asn Ile Lys Ile Ile Arg Asn Gly Pro Cys Gly

50 55 60

gly

65

<210> 24

<211> 65

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial

<400> 24

Gly Pro Gln Phe Gly Leu Phe Ser Lys Tyr Arg Thr Pro Asn Cys Ala

1 5 10 15

Asp His Ala Gly Met Ala Cys Val Ala Leu Tyr Glu Pro Val Cys Gly

20 25 30

Ser Asp Met Ser Thr Tyr Glu Asn Glu Cys Val Leu Cys Met Lys Ile

35 40 45

Arg Glu Gly Gly His Asn Ile Lys Ile Ile Arg Asn Gly Pro Cys Gly

50 55 60

gly

65

<210> 25

<211> 65

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial

<400> 25

Ser Pro Gln Phe Gly Leu Phe Ser Lys Tyr Arg Thr Pro Asn Cys Ala

1 5 10 15

Asp His Ala Gly Met Ala Cys Val Ala Leu Tyr Glu Pro Val Cys Gly

20 25 30

Ser Asp Met Ser Thr Tyr Glu Asn Glu Cys Val Leu Cys Met Lys Ile

35 40 45

Arg Glu Gly Gly His Asn Ile Lys Ile Ile Arg Asn Gly Pro Cys Gly

50 55 60

gly

65

<210> 26

<211> 65

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial

<400> 26

Glu Pro Gln Phe Gly Leu Phe Ser Lys Tyr Arg Thr Pro Asn Cys Ala

1 5 10 15

Asp His Ala Gly Met Ala Cys Val Ala Leu Tyr Glu Pro Val Cys Gly

20 25 30

Ser Asp Met Ser Thr Tyr Glu Asn Glu Cys Val Leu Cys Met Lys Ile

35 40 45

Arg Glu Gly Gly His Asn Ile Lys Ile Ile Arg Asn Gly Pro Cys Gly

50 55 60

gly

65

<210> 27

<211> 67

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial

<400> 27

Ser Leu Ile Pro Gln Phe Gly Leu Phe Ser Lys Tyr Arg Thr Pro Asn

1 5 10 15

Cys Ala Asp His Ala Gly Met Ala Cys Val Ala Leu Tyr Glu Pro Val

20 25 30

Cys Gly Ser Asp Met Ser Thr Tyr Glu Asn Glu Cys Val Leu Cys Met

35 40 45

Lys Ile Arg Glu Gly Gly His Asn Ile Lys Ile Ile Arg Asn Gly Pro

50 55 60

Cys Gly Gly

65

<210> 28

<211> 65

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial

<400> 28

Gly Pro Gln Phe Gly Leu Phe Ser Lys Tyr Arg Thr Pro Asn Cys Ala

1 5 10 15

Asp Phe Asp Gly Met Ala Cys Tyr Ala Phe Tyr Glu Pro Val Cys Gly

20 25 30

Ser Asp Met Ser Thr Tyr Ala Asn Glu Cys Thr Leu Cys Met Lys Ile

35 40 45

Arg Glu Gly Gly His Asn Ile Lys Ile Ile Arg Asn Gly Pro Cys Gly

50 55 60

gly

65

<210> 29

<211> 65

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial

<400> 29

Gly Pro Gln Phe Gly Leu Phe Ser Lys Tyr Arg Thr Pro Asn Cys Ala

1 5 10 15

Gln His Glu Gly Met Ala Cys Tyr Ala Leu Tyr Glu Pro Val Cys Gly

20 25 30

Ser Asp Met Ser Thr Tyr Ala Asn Glu Cys Thr Leu Cys Met Lys Ile

35 40 45

Arg Glu Gly Gly His Asn Ile Lys Ile Ile Arg Asn Gly Pro Cys Gly

50 55 60

gly

65

<210> 30

<211> 63

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> any amino acid

<220>

<221> MISC_FEATURE

<222> (16)..(19)

<223> any amino acid

<220>

<221> MISC_FEATURE

<222> (21)..(21)

<223> any amino acid

<220>

<221> MISC_FEATURE

<222> (24)..(24)

<223> any amino acid

<220>

<221> MISC_FEATURE

<222> (26)..(26)

<223> any amino acid

<220>

<221> MISC_FEATURE

<222> (27)..(27)

<223> any amino acid

<220>

<221> MISC_FEATURE

<222> (39)..(39)

<223> any amino acid

<220>

<221> MISC_FEATURE

<222> (43)..(43)

<223> any amino acid

<400> 30

Xaa Pro Gln Phe Gly Leu Phe Ser Lys Tyr Arg Thr Pro Asn Cys Xaa

1 5 10 15

Xaa Xaa Xaa Gly Xaa Ala Cys Xaa Ala Xaa Xaa Glu Pro Val Cys Gly

20 25 30

Ser Asp Met Ser Thr Tyr Xaa Asn Glu Cys Xaa Leu Cys Met Lys Ile

35 40 45

Arg Glu Gly Gly His Asn Ile Lys Ile Ile Arg Asn Gly Pro Cys

50 55 60

<210> 31

<211> 39

<212> PROTEIN

<213> Artificial sequence

<220>

<223> synthesized peptide

<400> 31

Gly Ser Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His

1 5 10 15

Met Asp Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met Ala

20 25 30

Asp Ile Gly Ser Ala Asn Ser

35

<210> 32

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> synthesized peptide

<400> 32

Gly Ala Ser Ala Ala Ala

15

<210> 33

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 33

aaaagaattc tgatccgcag tttggtctgt ttag 34

<210> 34

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 34

aaaactcgag ttatgcggcc gcagacgcgc cgcacggacc 40

<210> 35

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 35

aaaaggatcc ctggacaaac gtgatccgca gtttggtctg tttag 45

<210> 36

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 36

aaaactcgag ttagccgccg cacggaccat tgcgaataat ttta 44

<210> 37

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 37

aaacatatgg ggcaggaaga tcccaacagt ttgc 34

<210> 38

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 38

aaactcgagt ttggcctgtc ggtcatggga ctc 33

<210> 39

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 39

aaaagaattc gccaccatgc agattcctag agccg 35

<210> 40

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 40

aaaactcgag tcagtggtga tggtggtggt ggccgg 36

<210> 41

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 41

cttgtcgtca tcgtccttgt agtcgccggg gtcgatttcc tc 42

<210> 42

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 42

gcgactacaa ggacgatgac gacaagcacc accaccatca tcac 44

<210> 43

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 43

aaaaactcga gctagtgatg atggtggtgg tgcttgtcgt c 41

<210> 44

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 44

ccgcagtttg gtctgtttag caaatatcgt accccgaatt gt 42

<210> 45

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 45

gccataccag catggtccgc acaattcggg gtacgatatt tgc 43

<210> 46

<211> 40

<212> DNA

<213> Artificial sequence

<400> 46

gcggaccatg ctggtatggc atgtgttgct ctgtatgaac 40

<210> 47

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 47

aaaactcgag ttagccgccg cacggaccat tgcgaataa 39

<210> 48

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 48

aaaaggatcc ctggacaaac gtgatccgca gtttggtctg tttag 45

<210> 49

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 49

aaaaggatcc ctggacaaac gtggcccgca gtttggtctg tttag 45

<210> 50

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 50

aaaaggatcc ctggacaaac gtagcccgca gtttggtctg tttag 45

<210> 51

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 51

aaaaggatcc ctggacaaac gtgaaccgca gtttggtctg tttag 45

<210> 52

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 52

aaaaggatcc ctggacaaac gtagcctgat tccgcagttt ggtctgttta g 51

<210> 53

<211> 458

<212> PROTEIN

<213> Homo sapiens

<400> 53

Gln Leu Ser Arg Ala Gly Arg Ser Ala Pro Leu Ala Ala Gly Cys Pro

1 5 10 15

Asp Arg Cys Glu Pro Ala Arg Cys Pro Pro Gln Pro Glu His Cys Glu

20 25 30

Gly Gly Arg Ala Arg Asp Ala Cys Gly Cys Cys Glu Val Cys Gly Ala

35 40 45

Pro Glu Gly Ala Ala Cys Gly Leu Gln Glu Gly Pro Cys Gly Glu Gly

50 55 60

Leu Gln Cys Val Val Pro Phe Gly Val Pro Ala Ser Ala Thr Val Arg

65 70 75 80

Arg Arg Ala Gln Ala Gly Leu Cys Val Cys Ala Ser Ser Glu Pro Val

85 90 95

Cys Gly Ser Asp Ala Asn Thr Tyr Ala Asn Leu Cys Gln Leu Arg Ala

100 105 110

Ala Ser Arg Arg Ser Glu Arg Leu His Arg Pro Pro Val Ile Val Leu

115 120 125

Gln Arg Gly Ala Cys Gly Gln Gly Gln Glu Asp Pro Asn Ser Leu Arg

130 135 140

His Lys Tyr Asn Phe Ile Ala Asp Val Val Glu Lys Ile Ala Pro Ala

145 150 155 160

Val Val His Ile Glu Leu Phe Arg Lys Leu Pro Phe Ser Lys Arg Glu

165 170 175

Val Pro Val Ala Ser Gly Ser Gly Phe Ile Val Ser Glu Asp Gly Leu

180 185 190

Ile Val Thr Asn Ala His Val Val Thr Asn Lys His Arg Val Lys Val

195 200 205

Glu Leu Lys Asn Gly Ala Thr Tyr Glu Ala Lys Ile Lys Asp Val Asp

210 215 220

Glu Lys Ala Asp Ile Ala Leu Ile Lys Ile Asp His Gln Gly Lys Leu

225 230 235 240

Pro Val Leu Leu Leu Gly Arg Ser Ser Glu Leu Arg Pro Gly Glu Phe

245 250 255

Val Val Ala Ile Gly Ser Pro Phe Ser Leu Gln Asn Thr Val Thr Thr

260 265 270

Gly Ile Val Ser Thr Thr Gln Arg Gly Gly Lys Glu Leu Gly Leu Arg

275 280 285

Asn Ser Asp Met Asp Tyr Ile Gln Thr Asp Ala Ile Ile Asn Tyr Gly

290 295 300

Asn Ser Gly Gly Pro Leu Val Asn Leu Asp Gly Glu Val Ile Gly Ile

305 310 315 320

Asn Thr Leu Lys Val Thr Ala Gly Ile Ser Phe Ala Ile Pro Ser Asp

325 330 335

Lys Ile Lys Lys Phe Leu Thr Glu Ser His Asp Arg Gln Ala Lys Gly

340 345 350

Lys Ala Ile Thr Lys Lys Lys Tyr Ile Gly Ile Arg Met Met Ser Leu

355 360 365

Thr Ser Ser Lys Ala Lys Glu Leu Lys Asp Arg His Arg Asp Phe Pro

370 375 380

Asp Val Ile Ser Gly Ala Tyr Ile Ile Glu Val Ile Pro Asp Thr Pro

385 390 395 400

Ala Glu Ala Gly Gly Leu Lys Glu Asn Asp Val Ile Ile Ser Ile Asn

405 410 415

Gly Gln Ser Val Val Ser Ala Asn Asp Val Ser Asp Val Ile Lys Arg

420 425 430

Glu Ser Thr Leu Asn Met Val Val Arg Arg Gly Asn Glu Asp Ile Met

435 440 445

Ile Thr Val Ile Pro Glu Glu Ile Asp Pro

450 455

<210> 54

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> synthesized peptide

<220>

<221> BINDING

<222> (1)..(1)

<223> N-(4-methylcoumaryl-7-amide)isoleucine

<220>

<221> BINDING

<222> (10)..(10)

<223> N-epsilon-(2,4-dinitrophenyl)lysine

<400> 54

Xaa Arg Arg Val Ser Tyr Ser Phe Lys Xaa

1 5 10

<210> 55

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 55

ccatcatcaa ctacggcaac gcgggcggac ccctcgtgaa cc 42

<210> 56

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 56

ggttcacgag gggtccgcc gcgttgccgt agttgatgat gg 42z

<---

Claims (32)

1. A mutant SPINK2 peptide that contains the amino acid sequence shown in any of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23-29 (Fig. 15, 17, 19 , 21, 23, 25, 27, 29, 31, 33 and 35-41), and inhibits the protease activity of human HTRA1. 2. The peptide according to claim 1, which contains an amino acid sequence obtained by linking a peptide bond of one to three amino acids to the amino-terminal portion of the amino acid sequence shown in any of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17 , 19, 21 and 23-29 (Figs. 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 and 35-41). 3. The peptide according to claim 1, which contains an amino acid sequence obtained by linking a peptide bond of one or two amino acids to the carboxy-terminal portion of the amino acid sequence shown in any of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17 , 19, 21 and 23-29 (Figs. 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 and 35-41). 4. The peptide according to claim 1, which has a three-dimensional structure, characterized by the presence of three disulfide bonds and includes a loop structure, α-helix and β-sheet. 5. A polynucleotide for use in the treatment or prevention of a disease associated with HTRA1, comprising a nucleotide sequence encoding an amino acid sequence contained in a peptide according to any one of claims 1 to 4. 6. Vector for use in the treatment or prevention of a disease associated with HTRA1, containing a polynucleotide according to claim 5. 7. A cell for use in the treatment or prevention of a disease associated with HTRA1, containing a polynucleotide according to claim 5 or a vector according to claim 6, or producing a peptide according to any one of claims 1-4. 8. A method for producing a mutant SPINK2 peptide that inhibits the protease activity of HTRA1, comprising the following steps (i) and (ii): (i) culturing the cell according to claim 7 and (ii) isolating the mutant SPINK2 peptide from the culture. 9. A mutant SPINK2 peptide that inhibits HTRA1 protease activity obtained by the method of claim 8, wherein said peptide contains the amino acid sequence shown in any of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23-29 (FIGS. 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 and 35-41). 10. A conjugate for the treatment or prevention of a disease associated with HTRA1, containing a peptide according to any one of claims 1 to 4 and 9 associated with an additional drug that exhibits or enhances pharmacological activity. 11. The conjugate of claim 10 which is a polypeptide. 12. Pharmaceutical composition for the treatment or prevention of a disease associated with HTRA1, containing a therapeutically or prophylactically effective amount of a peptide according to any one of claims 1-4 and 9, a polynucleotide according to claim 5, a vector according to claim 6, a cell according to claim 7 and /or conjugate according to claim 10 or 11. 13. The pharmaceutical composition according to claim 12, wherein the disease associated with HTRA1 is one, two or more diseases selected from the group consisting of wet age-related macular degeneration, dry age-related macular degeneration, geographic atrophy, diabetic retinopathy, retinopathy of prematurity , polypoid choroidal vasculopathy, rheumatoid arthritis and osteoarthritis. 14. Pharmaceutical composition according to claim 12 or 13, which contains one, two or more additional drugs. 15. Pharmaceutical composition according to claim 12 or 13, which is used in combination with one, two or more additional drugs. 16. The pharmaceutical composition according to claim 12 or 13, which is a retinal protective agent. 17. Pharmaceutical composition according to claim 12 or 13 for the treatment or prevention of the dry form of age-related macular degeneration. 18. Pharmaceutical composition according to claim 12 or 13 for the treatment or prevention of the wet form of age-related macular degeneration. 19. The use of a pharmaceutical composition according to claim 12 for the treatment and prevention of a disease associated with HTRA1. 20. Use according to claim 19, wherein the disease associated with HTRA1 is one, two or more diseases selected from the group consisting of wet age-related macular degeneration, dry age-related macular degeneration, geographic atrophy, diabetic retinopathy, retinopathy of prematurity, polypoid choroidal vasculopathy, rheumatoid arthritis and osteoarthritis. 21. Use according to claim 19, wherein the pharmaceutical composition contains one, two or more additional drugs. 22. Use according to claim 19, where the pharmaceutical composition is used in combination with one, two or more additional drugs. 23. Use according to any one of claims 19-22, wherein the pharmaceutical composition is a retinal protective agent. 24. The use according to claim 19 or 20, which is intended for the treatment or prevention of the dry form of age-related macular degeneration. 25. The use according to claim 19 or 20, which is for the treatment or prevention of the wet form of age-related macular degeneration. 26. A conjugate for delivering a peptide according to any one of claims 1 to 4 and 9 to a specific cell compartment, comprising a peptide according to any one of claims 1 to 4 and 9 linked to a signal sequence present at the N-terminus of said peptide or added to N -end of said peptide. 27. A peptide affinity purification conjugate according to any one of claims 1-4 and 9, containing a peptide according to any one of claims 1-4 and 9 associated with an affinity tag. 28. A conjugate for detecting HTRA1, containing a peptide according to any one of claims 1-4 and 9, associated with an enzymatic label, radioactive label, color label, fluorescent label, dye label, luminescent label, hapten, digoxigenin, biotin, metal complex, metal or colloidal gold. 29. A conjugate for the treatment or prevention of a disease associated with HTRA1, comprising a peptide according to any one of claims 1-4 and 9, associated with a molecule that exhibits one or more of a binding affinity, an inhibitory activity, an antagonistic activity, and an agonistic activity against the molecule -target other than HTRA1. 30. A conjugate for the treatment or prevention of a disease associated with HTRA1, comprising a peptide according to any one of claims 1 to 4 and 9 linked to a molecule that improves the blood half-life of said peptide.

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